Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Oct 15.
Published in final edited form as: J Neuroimmunol. 2021 Jul 31;359:577675. doi: 10.1016/j.jneuroim.2021.577675

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

Emma E Kraus 1, Laura Kakuk-Atkins 1, Marissa F Farinas 2, Matthew Jeffers 2, Amy E Lovett-Racke 3, Yuhong Yang 1,3
PMCID: PMC8435019  NIHMSID: NIHMS1733429  PMID: 34403862

Abstract

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.

Keywords: Multiple Sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), Forkhead box O 1 (FoxO1), AS1842856, T effector cells, T regulatory cells, Central Nervous System (CNS)

1. Introduction

Multiple Sclerosis (MS) is an immune-mediated central nervous system (CNS) disease characterized by neuroinflammation, demyelination, and neuronal degeneration. MS is the leading cause of non-traumatic neurologic disability in young adults and affects over 1 million people in US (Wallin et al., 2019). Myelin-specific CD4 T effector cells (Teff), Th1 and Th17 cells, drive the formation of acute inflammatory demyelinating lesions and clinical relapses in experimental autoimmune encephalomyelitis (EAE) model of MS and have been implicated in MS pathogenesis (Frohman et al., 2006, McFarland and Martin, 2007). Early studies demonstrated Th1 cells that express key transcription factor T-bet and signature cytokine IFNγ to be highly encephalitogenic in EAE (Lovett-Racke et al., 2011). Subsequent studies have identified Th17 cells as another encephalitogenic CD4 Teff population in EAE. Th17 cells display a high degree of plasticity upon antigen reencounter, depending on the inflammatory milieu in the microenvironment (Guery and Hugues, 2015). TGFβ/IL-6 induces the development of Th17 cells that express the transcription factor RORγt and cytokine IL-17. However, they are not encephalitogenic in EAE adoptive transfer studies (Das et al., 2009, Ghoreschi et al., 2010, Yang et al., 2009). In a microenvironment that is rich in IL-12 and/or IL-23, non-encephalitogenic RORγt+ Th17 cells may transdifferentiate into RORγt+ T-bet+ Th1-like cells and further convert into RORγt−T-bet+Th1 cells, both of which are highly pathogenic in MS/EAE and other autoimmune diseases (Kamali et al., 2019, Kebir et al., 2009, Nistala et al., 2010, van Langelaar et al., 2018).

Forkhead box O 1 (FoxO1) is a conserved effector molecule in PI3k/Akt signaling that regulates CD4 T cell development and function. The role of FoxO1 in regulating myelin-specific Th1 in CNS autoimmunity has not been well-characterized. Although FoxO1 has been shown to suppress TGFβ/IL-6-induced differentiation of naïve CD4 T cells into RORγt+Th17 cells (Laine et al., 2015), its role in regulating Th17 transdifferentiation is unclear. FoxO1 plays a critical role in maintaining naive T cell quiescence and survival. As several FoxO-targeted genes are important regulators of naïve T cell trafficking and functions (Hedrick et al., 2012, Kerdiles et al., 2009, Kerdiles et al., 2010), FoxO1 deficiency may alter the quiescence of naïve CD4 T cells. Therefore, we took a pharmacological approach to characterize the potential role of FoxO1 signaling in regulating autoreactive CD4 Teff and Treg cells. AS1842856 is a small molecule compound that binds FoxO1 and inhibits FoxO1 transactivation (Nagashima et al., 2010). AS1842856 has been used extensively to determine the role of FoxO1 in regulating glucose metabolism, pulmonary hypertension, adipocyte differentiation and cancer development (Diep et al., 2013, Nagashima, Shigematsu, 2010, Savai et al., 2014, Wang et al., 2018, Zou et al., 2014). To understand the potential role of FoxO1 signaling in CNS autoimmunity, we determined the effects of AS1842856 in regulating myelin-specific Th1 and Th17 cells, and the transcriptional balance of T-bet and Foxp3 in myelin-specific CD4 T cells from EAE mice. The effects of AS1842856 in regulating the encephalitogenicity of myelin-specific T cells and the expansion of human Th1 cells from MS patients were also characterized. Furthermore, we characterized the potential role of FoxO1 in mediating PD-1 signaling in CD4 T cells, critical for regulating Teff and Treg cells.

2. Material and Methods

2.1. Animals

C57BL/6, SJL/J mice and TCR transgenic 2D2 mice that are specific for myelin oligodendrocyte glycoprotein (MOG) 35–55 were purchased from the Jackson Laboratory. B10.PL mice transgenic for the myelin basic protein (MBP) Ac1-11-specific TCR Vα2.3 or Vβ8.2 (Goverman et al., 1993) were bred in a pathogen-free animal facility at Ohio State University. Age 8–12 wks male and female C57BL/6 and B10.PL mice and female SJL/J mice were used for EAE studies as male SJL/J mice are resistant to EAE induction. Animal protocols were approved by the OSU Institutional Animal Care and Use Committee.

2.2. Human subjects

Peripheral blood mononuclear cells (PBMCs) were isolated from 6 MS patients, including 4 females and 2 males. One patient had relapsing-remitting MS (RRMS), 1 patient had secondary progressive MS (SPMS), and 4 patients had primary progressive MS (PPMS). Three patients were treat-naïve for disease-modifying therapies, 1 patient was treated with Avonex, and 2 patients were treated with Copaxone. Patient age ranges from 36–56 years, with a mean age of 47.8 years. Disease duration ranges from 5 months – 20 years, with a mean disease duration of 7.9 years. Blood was obtained by leukapheresis from MS patients after IRB approval and informed consent. PBMCs were isolated over a Ficoll gradient and stored in liquid nitrogen until use.

2.3. EAE induction

Immunization: EAE was induced in 8–10 week old female SJL/J mice by subcutaneously injection (s.c.) over four sites in the flank with 200 μg proteolipid protein (PLP) 139–151 respectively (C S bio) in an emulsion with CFA (Difco). 200 ng pertussis toxin (List) per mouse in PBS was injected intraperitoneally (i.p.) at the time of immunization.

Adoptive transfer: Splenocytes from naive 5–10 week-old B10.PL Vα2.3/Vβ8.2 TCR transgenic mice or immunized SJL/J mice (days 14–21 post-immunization) were activated with 10 μg/ml of MBP Ac1-11 or PLP 139–151 plus IL-12/IL-23 and AS1842856 (or DMSO) for 3 days. Then the cells were washed with PBS and 5–10 ×106 cells were injected i.p. into naive B10.PL mice or SJL/J mice.

Mice were scored on scale of 0 to 6: 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind limb weakness; 3, severe hind limb weakness; 4, complete hind limb paralysis; 5 quadriplegia or premoribund state; and 6, death.

2.4. In vitro culture of draining lymph node cells from EAE mice

Draining lymph node cells (dLNs) were prepared from immunized SJL/J mice or C57BL/6 mice around day 9 post immunization and activated in 24-well plates at 4–8×106 cells/well with of PLP 139–151 (2 μg/ml) or MOG35–55 (10 μg/ml), plus IL-23 (25 ng/ml) or IL-12 (0.5 ng/ml), in the presence of AS1842856 (0.1 μM) (Selleckchem). DMSO was used as vehicle control.

2.5. In vitro induction and expansion of Th1 and iTreg cells

Naïve CD4 T cells were purified from splenocytes of WT mice using Miltenyi naïve CD4 isolation kit. For Th1 induction in Figure 1A, naïve CD4 T cells were cultured on 24-well plates coated with 1 μg/ml of αCD3/CD28 (Biolegend) plus IL-12 (0.5 ng/ml) and AS1842856 (or DMSO) for 72h. For iTreg induction in Figure 5, αCD3/CD28 (1 μg/ml) and recombinant mouse PD-L1-Ig chimera or human IgG1-Ig (10 μg/ml) (Biolegend) were used to coat plates. Naïve CD4 T cells were cultured on coated plates plus TGFβ (4 μg/ml) and AS1842856 (or DMSO) for 72h. For the expansion of human Th1 cells in Figure 4 C–D, PBMCs from treatment-naïve MS patients were plated in flasks for 2–4 hrs to remove adherent cells. The suspension cells were then collected and activated with plate-bound αCD3/CD28 plus IL-12 (0.5 ng/ml) and AS1842856 (0.1 μM) or DMSO for 3 days.

Figure 1.

Figure 1.

Open in a new tab

FoxO1 inhibition with AS1842856 suppresses Th1 differentiation and expansion. (A-F) Naïve CD4 T cells were activated with αCD3/CD28 plus IL-12 for 3 days, in the presence of different concentrations of AS1842856 or vehicle control DMSO. T-bet (A-B) and IFNγ (C-D) were analyzed by intracellular staining, gating on CD44+CD4 T cells, and compared with one-way ANOVA. (E-F) Cell viability was analyzed by flow cytometry, gating on CD4 T cells. The live CD4 T cells (the left three bars) and live IFNγ+CD4 T cells (the right three bars) in AS1842856-treated groups were normalized to the control group and compared with 1-way ANOVA. (G-J) dLNs from immunized SJL/J mice were isolated on day 9 post immunization and activated with PLP 139–151 alone or plus IL-12 for 3 (G-H) or 6 days (I-J), in the presence of AS1842856 (0.1 μM) or vehicle control DMSO. (G, I) IFNγ was analyzed by intracellular staining, gating on CD44+CD4 T cells, and compared with unpaired student’s t test. (H, J) Cell viability was analyzed by flow cytometry, gating on CD4 T cells, and compared with 1-way ANOVA. Plots represent mean ± SEM of 3 independent experiments. **P<0.01; ***p<0.001; ****P<0.0001.

Figure 5.

Figure 5.

Open in a new tab

FoxO1 inhibition regulates iTreg development. (A) Naive CD4 T cells were activated with αCD3/CD28 plus TGFβ and different concentrations of AS1842856 or DMSO for 3 days. Foxp3 was determined by intracellular staining, gating on CD4+ T cells, and compared with one-way ANOVA. (B-D) Naïve CD4 T cells from WT/B6 mice were activated with αCD3/CD28 plus TGFβ and AS1842856 (0.1 μM) or DMSO for 3 days. Foxp3 and CD25 were measured by flow cytometry, gated on CD4+ cells (B). (C-D) The cells in (B) were then mixed with CFSE-labeled splenocytes from naïve 2D2 mice that are specific for MOG 35–55 at 1:4 ratio and activated with MOG35–55 for 5 days. The expression of CD44 (C) and CFSE (D) were determined by flow cytometry, gating on CFSE+ CD4+ cells, and compared with unpaired Student’s t-test (C). (E-F) Naïve CD4 T cells were activated with αCD3/CD28 plus TGFβ, in the presence of AS1842856 (0.1 μM), Rapamycin (10 nM), IC87114 (2 μM)/AS1842856 (0.1 μM), MK2206 (5 μM) /AS1842856 (0.1 μM) or DMSO for 3 days. Foxp3 and CD25 were measured by flow cytometry, gating on CD4+ cells, and compared with One-way ANOVA. (G-H) Naïve CD4 T cells were activated with plate-bound αCD3/CD28 and PD-L1-Ig or control-Ig plus TGFβ for 3 day, in the presence of AS1842856 (0.1 μM) or DMSO. Foxp3 expression was measured by intracellular staining, gating on CD4+ (G), and compared with 2-way ANOVA (H). Plots represent mean ± SEM of 3–6 independent experiments. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

Figure 4.

Figure 4.

Open in a new tab

FoxO1 inhibition suppresses T cell encephalitogenicity and the expansion of Th1 cells of MS patients. (A) Splenocytes from naive TCRαβ transgenic mice that are specific for MBP Ac1-11 were activated with MBP Ac1-11 and IL-12/IL-23 for 3 days in the presence of AS1842856 (0.1 μM). DMSO was used as vehicle control. At the end of culture, the cells were injected i.p. into naive B10 PL recipient mice. EAE was monitored in recipient mice. Disease incidence (sick mice/total mice) is indicated in parentheses. A statistically significant difference was considered to be P < 0.05, as determined by Mann-Whitney U-test. (B) dLNs from SJL/J mice that were immunized with PLP 139–151 were activated with PLP 139–151 plus IL-12/IL-23 for 3 days in the presence of AS1842856 or DMSO (0.1 μM). Then the cells were injected i.p. into naive SJL/J recipient mice. EAE development was monitored and compared by Mann-Whitney U-test. (C-G) PBMCs from treatment-naïve MS patients (n=6) were activated with plate-bound αCD3/CD28 plus IL-12 for 3 days, in the presence of AS1842856 (0.1 μM) or DMSO. CD45RA, IFNγ and T-bet expression was determined by flow cytometry, gating on CD4 cells, and compared with Wilcoxon matched-pairs signed rank test (E-G). *P<0.05. ****P<0.0001.

2.6. CFSE-based suppression assay

CD4 T cells from WT/B6 mice were activated on 24-well plates with plate-bound αCD3/CD28 (1 μg/ml) plus TGFβ (4 ng/ml) and AS1842856 (or DMSO) for 72h. Meanwhile, splenocytes from naïve TCR transgenic 2D2 mice that are specific for MOG 35–55 were labelled using CellTraceTM CFSE Cell Proliferation Kit (ThermoFisher Scientific). The iTreg cultured cells were washed and mixed with the CSFE-labelled splenocytes from 2D2 mice at 1:4 ratio in the presence of 10 μg/ml MOG 35–55 peptide for 4–5 days. The CFSE-labelled CD4+ Teff cells were evaluated using flow cytometry.

2.7. Flow cytometric analysis

Antibody (Ab) staining was performed to evaluate the expression of surface markers, transcription factors (T-bet, RORγt and Foxp3) and cytokines (IFNγ and IL-17) in CD4 T cells as described previously (Yang, Weiner, 2009). Briefly, the cells were first incubated with Abs to the cell-surface markers for 30’ at 4°C, followed by treatment with Cytofix/Cytoperm solution from ebioscience (for T-bet, RORγt and Foxp3) for 1h or BD Bioscience (for IFNγ and IL-17) for 20’–40’. Then cells were stained for transcription factors (T-bet, RORγt and Foxp3) or intracellular cytokines (IFNγ or IL-17) for 30’. Approximately 100,000 live cell events were acquired on a FACSCantoII and analyzed using FlowJo software (Tree Star, Inc.). PE-αIL-17, APC-αIFNγ, Pacific Blue-αT-bet, AF647-αFoxp3, FITC-αCD4, APC-αCD4, Pacific Blue-αCD44 and PE-Cy7-αCD25 were purchased from Biolegend. Fixable Viability Dye eFluor™ 780 and PE-αRORγt were purchased from eBioscience.

2.8. Statistics

GraphPad software (GraphPad Prism Software, Inc., San Diego, CA, USA) was utilized for statistical analysis. Quantitated flow data comparisons were performed using two-tailed unpaired Student’s t-test with two groups, one-way ANOVA with three or more groups or twoway ANOVA in Figure 2 G–H and Figure 3D. Mann-Whitney U-test was used to compare EAE data in Figure 4 A–B. Wilcoxon matched-pairs signed rank test was used to compare flow data in Figure 4 D. Differences with p<0.05 were considered significant.

Figure 2.

Figure 2.

Open in a new tab

FoxO1 inhibition suppresses the transdifferentiation of myelin-specific Th17 cells. dLNs from immunized SJL/J mice were isolated on day 9 post immunization and activated with PLP 139–151 (A, D), or plus IL-12 (B, E) or IL-23 (C, F), in the presence of AS1842856 (0.1 μM) or vehicle control DMSO for 3–6 days. T-bet and RORγt were analyzed by intracellular staining on day 3 (A-C) and day 6 (D-F), gating on CD44+CD4+ T cells. Unpaired student’s t test was used to compare the difference between the groups treated with AS1842856 (Squares) and vehicle control (Dots). % of CD4 T cells differentially express RORγt and T-bet in the groups treated with IL-12/DMSO or IL-23/DMSO was compared to the group treated with PLP/DMSO with 2-way ANOVA on day 3 (G) and day 6 (H). (I-J) Cell viability was analyzed by flow cytometry at day 3 (I) and day 6 (J), gating on CD4 T cells, and compared with 1-way ANOVA. Plots represent mean ± SEM of 3 independent experiments. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

Figure 3.

Figure 3.

Open in a new tab

FoxO1 inhibition shifts transcriptional balance of T-bet and Foxp3 in autoreactive CD4 T cells. dLNs from immunized SJL/J mice were activated with PLP 139–151 (A), plus IL-12 (B) or IL-23 (C), in the presence of AS1842856 (0.1 μM) or vehicle control DMSO for 3 days. Foxp3 and T-bet were analyzed by intracellular staining, gating on CD44+CD4 T cells. Unpaired student’s t test was used to compare the difference between the group treated with AS1842856 and vehicle control. % of CD4 T cells differentially express Foxp3 and T-bet in the group treated with IL-12/DMSO or IL-23/DMSO was compared to the group treated with PLP/DMSO with 2-way ANOVA (D). Plots represent mean ± SEM of 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001; **** P<0.0001.

3. Results:

3.1. FoxO1 inhibition with AS1842856 suppresses Th1 differentiation and expansion

Th1 cells are highly encephalitogenic in EAE and implicated in MS pathogenesis. To understand the potential role of FoxO1 signaling in CNS autoimmunity, we first determined whether FoxO1 inhibition with AS1842856 affects the differentiation of naïve CD4 T cells into Th1 cells. FoxO1 binds to the consensus FoxO binding sites in the promoter region of target genes, subsequently activating gene expression (Onuma et al., 2006). AS1842856 has been shown to selectively and potently repress Foxo1-mediated promoter activity in a dose-dependent manner using luciferase reporter assays (17). AS1842856 has been widely used to characterize the potential roles of FoxO1 signaling in various physiological and pathological conditions, including glucose metabolism, pulmonary hypertension, adipocyte differentiation and cancer development (Diep, Charles, 2013, Nagashima, Shigematsu, 2010, Savai, Al-Tamari, 2014, Wang, Demir, 2018, Zou, Liu, 2014). However, it is potential role in Th1 cell differentiation and function, and CNS autoimmunity has not been characterized. AS1842856 potently blocks FoxO1 activity at 0.05–1 μM without showing significant cellular cytotoxicity (Nagashima, Shigematsu, 2010). Thus, AS1842856 was used at 0.05 and 0.1 μM during Th1 differentiation. Naive CD4 T cells were activated with αCD3/CD28 plus IL-12, in the presence of AS1842856 or vehicle control DMSO (Figure 1A–E). Th1 key transcription factor T-bet and signature cytokine IFNγ were analyzed. T-bet+ (Figure 1A–B) and IFNγ+ CD4 T cells (Figure 1C–D) were significantly lower in AS1842856-treated groups compared to the control group, suggesting FoxO1 inhibition with AS1842856 suppresses the differentiation of naïve CD4 T cells into Th1 cells. FoxO transcription factors are important regulators of cell quiescence and longevity. Therefore, we determined whether AS1842856 treatment alters cell death of treated CD4 cells, which may contribute to the suppressive effects on Th1 development (Figure 1E–F). Although total live CD4 T cells in AS1842856-treated groups were slightly lower than control group, the difference was not significant (Figure 1 F left panels). The IFNγ expressing live CD4 T cells were significantly lower in AS1842856-treated groups compared to control group (Figure 1F right panels), suggesting that cell death of AS1842856-treated cells is not be a major contributor of the decreased Th1 differentiation.

To determine whether FoxO1 inhibition alters the expansion of myelin-specific Th1 cells from EAE mice, dLNs from immunized SJL/J mice were activated in vitro with PLP 139–151 alone or plus IL-12 for 3–6 days, in the presence of AS1842856 or vehicle control DMSO (Figure 1G–J). There was no significant difference of IFNγ+ CD4 T cells between AS1842856 and the control group when activated with myelin antigen alone (Figure 1G, I, left panels). However, in the presence of IL-12, IFNγ+ Th1 cells were significantly lower in AS1842856 groups compared to control groups at both day 3 and day 6 post reactivation (Figure 1G, I, right panels), suggesting FoxO1 inhibition significantly suppresses IL-12-induced IFNγ expression of effector/memory myelin-specific Th1 cells from EAE mice. There is no significant difference of live CD4 T cells between AS1842856-treated groups and control groups (Figure 1H, J). These data illustrate the role of FoxO1 inhibition in suppressing Th1 differentiation and the expansion of myelin-specific Th1 cells.

3.2. FoxO1 inhibition suppresses the transdifferentiation of myelin-specific Th17 cells

Th17 cells are also a subset of CD4 Teff cells that can be encephalitogenic in EAE and implicated in MS pathogenesis (Lovett-Racke, Yang, 2011). Although TGFβ/IL-6 induces the differentiation of naïve CD4 T cells into Th17 cells that express transcription factor RORγt and cytokine IL-17, those TGFβ/IL-6-induced Th17 cells fail to induce EAE when injected into wild type recipient mice (Das, Ren, 2009, Ghoreschi, Laurence, 2010, Yang, Weiner, 2009). Upon antigen reencounter, RORγt+ Th17 cells may transdifferentiate into Th1-like Th17 cells that co-express RORγt and the Th1 key transcription factor T-bet (RORγt+ T-bet+), which are highly encephalitogenic in adoptive transfer, and may further convert into Th1 cells that do not express RORγt (RORγt− T-bet+) (Guery and Hugues, 2015, Kamali, Noorbakhsh, 2019, Kebir, Ifergan, 2009, Nistala, Adams, 2010, van Langelaar, van der Vuurst de Vries, 2018). While Th17 transdifferentiation may occur naturally, IL-12 and IL-23 promote Th17 transdifferentiation. mTORC1 has been shown to be important for Th17 transdifferentiation (Karmaus et al., 2019). As mTORC1 and FoxO1 are two conserved effector molecules of PI3K/Akt signaling, we determined whether FoxO1 plays a role in regulating the transdifferentiation of myelin-specific Th17 cells. dLN cells from EAE mice were reactivated in vitro with myelin antigen, or plus IL-12 or IL-23 for 3–6 days, in the presence of AS1842856 or vehicle control DMSO (Figure 2). Subpopulations of CD4 T cells differentially expressing RORγt and T-bet were analyzed to determine whether FoxO1 regulates specific subsets of Th1 or Th17 cells. CD4 T cells expressing RORγt, but not T-bet (RORγt+T-bet−), represent non-encephalitogenic Th17 cells, while encephalitogenic Th1-like Th17 cells are RORγt+ T-bet+. Th1 cells are RORγt− T-bet+. As shown in Figure 2, both Th1-like Th17 cells (RORγt+ T-bet+) and Th1 cells (RORγt− T-bet+) were significantly lower in AS1842856-treated groups compared to the control groups at day 3 and day 6 post reactivation (Figure 2A, D), suggesting FoxO1 inhibition suppresses the transdifferentiation of Th17 cells and the expansion of Th1 cells. Addition of IL-12 significantly increased the percentage of Th1 cells (RORγt− T-bet+) at day 3 and 6 post reactivation (Figure 2G–H). Addition of IL-23 significantly increased the percentage of Th1-like Th17 cells (RORγt+ T-bet+) and Th1 cells (RORγt− T-bet+) at day 3 and 6 post reactivation, compared to the group treated with myelin antigen alone (Figure 2 G–H). These data confirm that IL-12 and IL-23 promote Th1 expansion and Th17 transdifferentiation. In the presence of IL-12 or IL-23, both Th1-like Th17 cells (RORγt+ T-bet+) and Th1 cells (RORγt−T-bet+) were significantly lower in AS1842856-treated groups compared to the control groups at day 3 and day 6 post reactivation (Figure 2B, C, E, F). Non-encephalitogenic Th17 cells (RORγt+ T-bet−) were at similar levels in AS1842856-treated groups and the control groups at both time points. There is no significant difference of live CD4 T cells during Th17 transdifferentiation between AS1842856-treated groups and control groups at day 3 (Figure 2I) or day 6 (Figure 2J). These data suggest that FoxO1 inhibition significantly suppresses Th17 transdifferentiation and Th1 expansion of autoreactive CD4 T cells from EAE mice.

3.3. FoxO1 inhibition shifts transcriptional balance of T-bet and Foxp3 in autoreactive CD4 T cells

As Foxp3 expressing CD4 Tregs have the potential to suppress encephalitogenic Teff cells (Arellano et al., 2016), the balance between Tregs and encephalitogenic Teff cells may decide the outcome of inflammation and be critical for the progression of autoimmunity. As our data show that FoxO1 inhibition suppresses T-bet+ Th1 cells as well as the transdifferentiation of Th17 cells into encephalitogenic RORγt+ T-bet+ Th1-like cells (Figure 1–2), we determined whether the transcriptional balance of T-bet: Foxp3 of myelin-specific CD4 T cells from EAE mice may be shifted by AS1842856. The expression of T-bet and Foxp3 were analyzed in myelin-specific CD4 T cells from EAE mice that were reactivated ex vivo with myelin antigen, in the presence of AS1842856 or vehicle control DMSO (Figure 3). Foxp3+ Tregs show functional plasticity and may adopt Teff phenotype under inflammatory conditions (Laurence et al., 2012, Sakaguchi et al., 2013, Yang et al., 2008). IFNγ expressing Foxp3+ Tregs have been identified in MS patients with reduced suppressive function (Dominguez-Villar et al., 2011). Thus, Foxp3+ T-bet+ cells may represent Tregs with reduced suppressive function while Foxp3+ T-bet− cells represent functional Tregs. T-bet+ Foxp3− cells represent encephalitogenic CD4 Teff cells, including Th1 and/or Th1-like Th17 cells. As shown in Figure 3, encephalitogenic Teff cells (T-bet+ Foxp3−) were significantly lower in the group treated with AS1842856 compared to the control group (Figure 4A), confirming FoxO1 inhibition suppresses encephalitogenic T-bet+ Teff cells. Meanwhile, functional Treg cells (Foxp3+ T-bet−) were significantly higher, while Tregs with reduced suppressive function (Foxp3+ T-bet+) were significantly lower in the group treated with AS1842856 compared to the control group (Figure 4A), suggesting AS1842856 may stabilize functional Tregs. Addition of IL-12 or IL-23 led to a significant increase of T-bet+ CD4 T cells, especially encephalitogenic T-bet+ Foxp3− Teff cells (Figure 4D), confirming the critical role of IL-12 and IL-23 in promoting T cell encephalitogenicity. In the presence of IL-12 or IL-23, AS1842856 treatment led to a significant decrease of encephalitogenic Teff (T-bet+ Foxp3−) cells and Tregs with reduced suppressive function (Foxp3+ T-bet+), while functional Treg (Foxp3+ T-bet−) cells were significantly higher in the groups treated with AS1842856 compared to the control groups (Figure 4B–C). These data indicate that FoxO1 inhibition shifts the transcriptional balance between Tregs and encephalitogenic Teff cells toward Tregs, potentially favoring the resolution of inflammation.

3.4. FoxO1 inhibition suppresses T cell encephalitogenicity and the expansion of Th1 cells of MS patients

To understand the impact of FoxO1 in CNS autoimmunity, we determined whether AS1842856 may affect T cell encephalitogenicity in EAE and the expansion of pathogenic Teff cells from MS patients. Although AS1842856 has been administered in vivo to diabetic mice for a short period of time, most studies were limited to in vitro studies as the plasma concentration of AS1842856 could not be detected 2 hrs after oral administration (Nagashima, Shigematsu, 2010), making it unsuitable for EAE studies in vivo. Therefore, we determined whether AS1842856 treatment in vitro altered the encephalitogenic potential of myelin-specific CD4 T cells following adoptive transfer. Adoptively transferred EAE study has been widely used to evaluate the encephalitogenicity of myelin-specific CD4 T cells in MS/EAE research. Splenocytes from MBP Ac1-11-specific TCR transgenic mice were activated with MBP Ac1-11 and IL-12/IL-23 for 3 days in the presence of 0.1 μM of AS1842856 or vehicle control DMSO, and injected into naive B10PL recipient mice (Figure 4A). EAE severity in the mice receiving AS1842856-treated myelin-specific CD4 T cells was significantly lower compared to the mice receiving control-treated cells (Figure 4A), indicating that FoxO1 inhibition suppresses the encephalitogenic potential of myelin-specific CD4 T cells. To make certain that the suppression of T cell encephalitogenicity by AS1842856 is not specific to this EAE model [MBP Ac1-11 or the major histocompatibility complex (MHC) (H-2u)], we performed a similar experiment by adoptively transferring splenocytes from immunized SJL/J mice (H-2s) that were activated with PLP 139–151 plus IL-12/IL-23 for 3 days ex vivo in the presence of AS1842856 or DMSO (Figure 4B). While the mice receiving vehicle control-treated cells developed EAE, none of the mice receiving AS1842856-treated cells developed EAE (Figure 4B), confirming that FoxO1 inhibition suppresses the encephalitogenicity of myelin-specific CD4 T cells. Using two different adoptive transfer EAE models, we are recapitulating the differentiation of naïve T cells into encephalitogenic T cells with FoxO1 inhibition (Figure 4A), and inhibition of FoxO1 in antigen-experienced CD4 T cells (Figure 4B) and analyzing the functional consequence in a pre-clinical model.

To understand the potential role of FoxO1 in regulating human Th1 cells, we determined whether AS1842856 may regulate the expansion of Th1 cells from MS patients. PBMCs were activated with αCD3/CD28 plus IL-12 for 3 days, in the presence of AS1842856 or vehicle control DMSO (Figure 4 C–D). The percentage of IFNγ+CD4 T cells were significantly lower in AS1842856-treated group compared to the control group (Figure 4E). The percentage of CD45RA− effector/memory CD4 T cells that express IFNγ (Figure 4F) and the percentage of T-bet+ IFNγ+ CD4 T cells (Figure 4G) were also significantly lower in AS1842856-treated group compared to the control group, demonstrating that AS1842856 suppresses the expansion of Th1 cells from MS patients. These data suggest that FoxO1 inhibition may limit pathogenic Th1 cells and provide therapeutic benefits in MS.

3.5. Inhibition of FoxO1 signaling regulates Treg development

The role of FoxO1 in regulating Treg development is controversial (Kerdiles, Stone, 2010, Luo et al., 2016, Ouyang et al., 2010). Early studies showed that FoxO1 deficiency in T cells impairs Treg development and leads to a mild autoimmune phenotype (Kerdiles, Stone, 2010, Ouyang, Beckett, 2010). However, unprimed FoxO1−/− CD4 T cells express relatively lower levels of TGFβRII compared to naïve FoxO1+/+ CD4 T cells from WT mice (Kerdiles, Stone, 2010), which may contribute to the impaired iTreg development of unprimed FoxO1−/− CD4 T cells since TGFβ signaling is instrumental in Treg development. A recent study revealed that FoxO1 signaling inhibits Treg-cell-mediated immune tolerance since Treg-specific expression of a constitutively-active FoxO1 mutant led to severe T-cell-mediated autoimmune diseases (Luo, Liao, 2016), suggesting FoxO1 inhibition may improve immune tolerance via promoting Tregs. Therefore, we determined whether FoxO1 inhibition with AS1842856 alters iTreg development. Purified naïve CD4 T cells were activated with αCD3/CD28 under iTreg differentiation condition in the presence of AS1842856 or vehicle control (Figure 5). Foxp3+ CD4 T cells were significantly higher in the groups treated with AS1842856 (0.025, 0.05 and 0.1 μM) compared to those treated with vehicle control, in a dose-dependent manner (Figure 5A), suggesting FoxO1 inhibition promotes iTreg development, which is in consistent with the previous study showing forced expression of FoxO1 impairs Treg development (Luo, Liao, 2016). To determine whether AS1842856-induced Foxp3+ Treg cells have suppressive function, AS1842856-treated cells (or control-treated cells) (Figure 5B) were mixed with CFSE-labeled CD4 T cells from TCR transgenic 2D2 mice that are specific for MOG 35–55, and activated with MOG 35–55 (Figure 5 B–D). The percentage of activated CD44+ CFSE+ CD4 Teff cells was significantly lower in the group cultured with AS1842856-cultured Tregs (Figure 5C), suggesting Treg cells cultured with AS1842856 are able to suppress the activation of myelin-specific CD4 Teff cells. CFSE proliferation assay showed the proliferation of CFSE+ CD4 T cells was notably lower in the group cultured with AS1842856-iTregs (Figure 5D), suggesting iTreg cells cultured with AS1842856 are able to suppress antigen-specific proliferation of myelin-specific CD4 Teff cells. The increased suppression of the activation and proliferation of myelin-specific CD4 Teff cells is likely due to the increased number of Foxp3+ Treg T cells in the AS1842856 group. These data illustrate that FoxO1 inhibition with AS1842856 promotes the development of functional iTreg cells, which may be able to suppress Teff cells and benefit the resolution of inflammation in autoimmunity.

Inhibition of mTORC1 signaling has been shown to promote iTreg development (Battaglia et al., 2006, Qu et al., 2007). Thus we compared the effects of mTORC1 inhibitor rapamycin and FoxO1 inhibitor AS1842856 in regulating iTreg development (Figure 5E). Inhibition of mTORC1 with rapamycin increased Foxp3+ iTreg cells. However, Foxp3+ iTreg cells were significantly higher in the AS1842856-treated group compared to the rapamycin-treated group (Figure 5E), suggesting FoxO1 is a major regulator of Foxp3 expression in CD4 T cells. Phosphorylation is critical for transcription factors, including FoxO1, to repress target gene expression (Langlet et al., 2017). FoxO1 is mainly phosphorylated by pAkt. To determine whether FoxO1 phosphorylation is important for FoxO1 regulation of Foxp3 expression, selective inhibitor of PI3k (IC87114) or Akt (MK2206) was used in combination with AS1842856 in iTreg cultures (Figure 5F). Foxp3+ iTreg cells were significantly lower in the groups treated with IC87114 (PI3k)/AS1842856 or MK2206 (Akt)/AS1842856 compared to the group treated with AS1842856 alone (Figure 5F), suggesting FoxO1 phosphorylation by PI3k/Akt signaling is required for AS1842856-induced Foxp3 expression in CD4 T cells.

Programmed cell death protein 1 (PD-1) is an important immune checkpoint that regulates immune tolerance and autoimmunity (Boettler and von Herrath, 2012, Carter et al., 2007, Juchem et al., 2018, Kroner et al., 2009, Latchman et al., 2004, Lee et al., 2012, Penaranda et al., 2012, Salama et al., 2003). PD-1 ligation enhances the development of TGFβ-induced Foxp3+ Treg cells (iTregs) (Francisco et al., 2009). As PD-1 ligation generally regulates PI3K/Akt signaling in CD4 T cells (Arasanz et al., 2017, Francisco, Salinas, 2009, Zuazo et al., 2017), we determined whether FoxO1 signaling plays a role in PD-1-induced Foxp3 expression in CD4 T cells. Naïve CD4 T cells were activated with plate-bound αCD3/CD28/PD-L1-Ig (or control-Ig) plus TGFβ for 3 days, in the presence of AS1842856 or vehicle control DMSO (Figure 5G). Foxp3+ iTregs were significantly higher in the group activated with PD-L1-Ig compared to the group activated with control-Ig (Figure 5 G left two panels), confirming PD-1 ligation promotes Foxp3 expression in CD4 T cells. However, the percentage of Foxp3+ iTregs in the groups activated with PD-L1-Ig and AS1842856 (Figure 5G upper row) is similar to the group activated with PD-L1-Ig and vehicle control DMSO (Figure 5G lower row). The fact that there are no additive effects of FoxO1 inhibition and PD-1 ligation suggests that PD-1 signaling pathway may overlap with FoxO1 signaling in regulating Foxp3 expression in CD4 T cells.

4. Discussion

Using a selective FoxO1 inhibitor AS1842856, we report that FoxO1 inhibition suppresses the differentiation and expansion of Th1 cells as well as the transdifferentiation of myelin-specific Th17 cells into Th1-like cells upon reactivation with myelin antigen. FoxO1 inhibition shifts the transcriptional balance of T-bet and Foxp3 of myelin-specific CD4 T cells from EAE mice toward Foxp3 upon reactivation with myelin antigen. FoxO1 inhibition also suppresses T cell encephalitogenicity in adoptively transferred EAE studies and the expansion of human Th1 cells from MS patients. In addition, inhibition of FoxO1 signaling impairs PD-1-induced Foxp3 expression.

As a conserved effector molecule of PI3K/Akt signaling pathway, FoxO1 plays a critical role in maintaining naive T cell quiescence and survival. As a result, some phenotypes observed in FoxO1 deficient mice or naïve T cells from FoxO1 deficient mice may reflect indirect effects of FoxO1 deficiency in T cells. Thus, it is important to use pharmacological approaches to determine the role of FoxO1 in regulating T cells and CNS autoimmunity. AS1842856 is a small-molecule compound that binds FoxO1 and potently inhibits its transactivation of target gene expression. Dose-dependent and selective inhibition of FoxO1-mediated transactivation by AS1842856 have been well-characterized by Nagashima et al using luciferase reporter assays (Nagashima, Shigematsu, 2010). Multiple studies have used AS1842856 to characterize the role of FoxO1 signaling in different physiological and pathological conditions. AS1842856 reduces glucose production in hepatic cells (Nagashima, Shigematsu, 2010), reproduces features of pulmonary hypertension in pulmonary artery smooth muscle cells (Savai, Al-Tamari, 2014) and suppresses adipocyte differentiation (Zou, Liu, 2014). AS1842856 also reduces leukemia load and prolongs survival in a preclinical model of BCP-ALL (Wang, Demir, 2018), inhibits progestin-induced p21 expression and blocks progestin-induced senescence in ovarian cancer (Diep, Charles, 2013). IC50 of AS1842856 to inhibit FoxO1 is 0.033 μM (Nagashima, Shigematsu, 2010). Although FoxO1, FoxO3 and FoxO4 share a high degree of sequence homology, FoxO1-mediated promoter activity was decreased by more than 70% while FoxO3- and FoxO4-mediated promoter activity by 3 and 20% respectively when AS1842856 was used at 0.1 μM (Nagashima, Shigematsu, 2010). FoxO3 deficiency in T cells does not alter Th17 development (Laine, Martin, 2015). FoxO4 deficient mice have no known phenotypic differences from WT mice (Hedrick, Hess Michelini, 2012). Therefore, the effects that we observed with AS1842856 appear due to inhibition of FoxO1. However, the plasma concentration of AS1842856 could not be detected 2 hours after orally administration (Nagashima, Shigematsu, 2010), which is not suitable for in vivo EAE studies. Since the data with AS1842856 supports the potential role of FoxO1 as a therapeutic target for CNS autoimmunity, backcrossing FoxO1fl/fl mice onto B6 and SJL backgrounds, which may be further crossed with tamoxifen-inducible CD4-cre mice could be used for the analysis of the effects of FoxO1-deletion in CD4 T cells after EAE development. Moreover, different approaches may be taken to improve the in vivo pharmacokinetic properties of small molecules. Prodrug strategies have shown great success in improving the properties of small molecules for therapeutic purposes (Rautio et al., 2018). We have recently taken the prodrug strategy and developed a novel prodrug STAT3 inhibitor LLL12b with significantly improved pharmacokinetic properties from the original small-molecule STAT3 inhibitor LLL12. While LLL12 doesn’t show any therapeutic effects in EAE studies in vivo because of the poor PK properties, therapeutic administration of LLL12b significantly suppresses disease development and progression in adoptively transferred, chronic, and relapsing-remitting EAE in vivo (Aqel et al., 2021). Additionally, encapsulating small molecules in microdroplets has also be explored to overcome the limitation of poor in vivo pharmacokinetic properties. LLL12-loaded microdroplets have shown promising efficacy for the treatment of cancer cells (Xu et al., 2017). Further investigation is needed to improve the pharmacokinetic properties of AS1842856 for evaluating the potential role of FoxO1 signaling in CNS autoimmunity in vivo.

The role of FoxO1 in regulating Th1 differentiation and expansion has not been well-characterized. Rao et al showed that CD8 T cells that were activated with IL-12 and transduced with FoxO1-RV had lower T-bet expression after restimulation, suggesting FoxO1 represses T-bet expression in type-1 CD8 T cells upon restimulation (Rao et al., 2012). However, the molecular mechanisms that regulate the development and function of type 1 CD8 T cells and Th1 CD4 T cells are different (Kallies and Good-Jacobson, 2017). T-bet is the key transcription factor driving IFNγ expression and Th1 differentiation of CD4 T cells. In contrast, T-bet-deficient CD8 T cells only show a mild reduction in IFNγ production, suggesting of different molecular mechanisms in regulating type 1 CD8 and Th1 CD4 T cell development. T-bet shares functions with transcription factors Eomesodermin (Eomes), and both factors cooperatively induce IFNγ production and expression of cytotoxic molecules in CD8 T cells. The detailed molecular mechanisms by which FoxO1 signaling regulates the expansion of Th1 CD4 T cells may be different from type-1 CD8 T cells. Furthermore, the role of FoxO1 in regulating the differentiation of naïve CD4 T cells into Th1 cells has not been characterized. Our data show that FoxO1 inhibition with AS1842856 leads to decreased T-bet and IFNγ expression, suggesting FoxO1 inhibition suppresses Th1 differentiation and expansion, although further investigation is needed to characterize the precise molecule mechanisms.

FoxO1 has been shown to suppress TGFβ/IL-6-induced differentiation of naïve CD4 T cells into RORγt+Th17 cells (Laine, Martin, 2015), however, its role in regulating Th17 transdifferentiation is not clear. mTORC1, another conserved effector molecules of PI3K/Akt signaling pathway, has been shown to be important for the Th17 transdifferentiation into encephalitogenic Th1-like cells (Karmaus, Chen, 2019). mTORC1 and FoxO1 may regulate each other’s expression directly or indirectly via feedback of the upper signaling molecules of PI3K/Akt pathway. pAkt positively regulates mTORC1 while mTORC1 suppresses pAkt activity through a negative feedback loop. Foxo1 is phosphorylated by pAkt, which leads to its nuclear exclusion and inactivation. Moreover, mTORC1 inhibition does not significantly alter IFNγ expression during T cell activation (Kurebayashi et al., 2012), making it unclear about the detailed mechanisms governing the expression of T-bet/IFNγ in Th1-like cells during Th17 transdifferentiation. We hypothesize that FoxO1 signaling may play a role in regulating Th17 transdifferentiation, potentially through altering IFNγ expression in Th17 cells. Our data support this hypothesis by showing that FoxO1 inhibition leads to decreased RORγt+ T-bet+ CD4 T population, suggesting FoxO1 signaling is important for Th17 transdifferentiation. However, further investigation is needed to dissect the detailed molecule mechanisms of PI3K/Akt signaling pathway in regulating Th17 transdifferentiation and T cell encephalitogenicity.

The role of FoxO1 in regulating Tregs is controversial. Early studies showed that FoxO1 deficiency in T cells impairs Treg development and leads to a mild autoimmune phenotype (Kerdiles, Stone, 2010, Ouyang, Beckett, 2010), suggesting that FoxO1 promotes Treg development. Transcriptionally active FoxO1 predominantly resides in the nucleus of naive T cells. After TCR activation, FoxO1 is phosphorylated, resulting its nuclear exclusion and termination of transcriptional activity. Several FoxO1-targeted genes are important regulators of naïve T cell trafficking and functions (Hedrick, Hess Michelini, 2012, Kerdiles, Beisner, 2009, Kerdiles, Stone, 2010). Unprimed FoxO1−/− CD4 T cells express relatively lower levels of TGFβRII compared to naïve FoxO1+/+ CD4 T cells from WT mice (Kerdiles, Stone, 2010), which may contribute to the impaired iTreg development of unprimed FoxO1−/− CD4 T cells. A subsequent study revealed an unexpected function of FoxO1 in inhibiting a Treg-cell-mediated immune tolerance (Luo, Liao, 2016). The differentiation of activated Tregs (aTregs) that are present in non-lymphoid tissue and critical for peripheral immune tolerance was shown to be associated with reduced FoxO1 expression and function. As a result, Treg-specific expression of a constitutively-active Akt-insensitive FoxO1 mutant led to severe T-cell-mediated autoimmune diseases, suggesting FoxO1 inhibition may improve immune tolerance via promoting Tregs. Our data using a selective FoxO1 inhibitor AS1842856 to treat CD4 T cells from naïve WT mice during iTreg differentiation suggest that FoxO1 inhibition promotes iTreg development from naïve CD4 T cells (Figure 5). Furthermore, suppression assays were performed to determine whether the Tregs induced in the presence of AS1842856 are active (Figure 5B–C). AS1842856-treated iTreg cultured cells show enhanced suppression of the proliferation of Teff cells, suggesting that Tregs in the AS1842856-treated group are functional and the increased Treg number may lead to better suppression of Teff proliferation. Further investigation of the suppressive function of sorted Tregs differentiated from naive CD4 T cells of GFP-Foxp3 reporter mice in the presence of AS1842856 (or vehicle control) will elucidate whether AS1842856 may alter the suppressive capacity of Tregs.

We also explored the role of FoxO1 signaling in mediating PD-1-induced Treg development, as PD-1 signaling generally downregulates PI3K/Akt signaling pathway (Arasanz, Gato-Canas, 2017, Zuazo, Gato-Canas, 2017). Our data show that FoxO1 inhibition impairs PD-1-induced Foxp3 expression (Figure 5G–H), suggesting a role of FoxO1 signaling in PD-1 regulation of Tregs. Although PD-1 ligation promotes iTreg development, the effects of PD-1 signaling in regulating suppressive function of Treg cells are not well-characterized. PD-L1 has been shown to enhance suppressive function of iTregs (Francisco, Salinas, 2009) while PD-1−/− Tregs lack suppressive function (Polanczyk et al., 2007), suggesting PD-1 ligation may promote Treg suppressive function. However, a recent publication shows that PD-1 deficiency in Treg cells leads to an activated phenotype and enhanced suppressive function, suggesting PD-1 signaling negatively regulate Treg function (Tan et al., 2021). As FoxO1 inhibition may promote Treg development, while impairing PD-1-mediated Treg development, the net effects of FoxO1 inhibition in regulating Treg development and function in the context of PD-1 signaling are not completely understood. Interestingly, similar scenario exists in cancer as both FoxO1 and PD-1 signaling are involved in various aspects of cancer (Deng et al., 2018, Hornsveld et al., 2018). While high dose expression of constitutively active Akt-insensitive FoxO1 expression suppressed aTregs and led to autoimmunity, low dose of Akt-insensitive FoxO1 expression depleted tumor-associated Treg cells, activated effector T cells, leading to inhibited tumor growth without inflicting autoimmunity (Luo, Liao, 2016), suggesting FoxO1 signaling pathway in Tregs can be titrated to regulate immune tolerance, potentially in ways benefiting cancer and/or autoimmunity. Further investigation that dissects the precise molecular mechanisms of FoxO1 signaling in PD-1 regulation of Treg development, migration and suppressive function may reveal innovative therapeutic targets for autoimmunity and cancer.

In addition to CD4 T cells, FoxO1 also plays a role in regulating antigen presenting cells (Cabrera-Ortega et al., 2017, Dong et al., 2015). FoxO1 deficiency in DCs decreases IL-12 produced by DCs in mucosal surfaces. Therefore, suppression of FoxO1 in DCs may contribute to the suppressive effects of AS1842856 in suppressing IFNγ and T-bet expression in myelin-specific CD4 T cells from EAE mice upon reactivation. However, the effects of AS1842856 in suppressing the differentiation of naïve CD4 T cells into Th1 cells (Figure 1) and mediating PD-1 signaling in CD4 T cells (Figure 5) were analyzed using purified naïve CD4 T cells activated with αCD3/CD28, which excludes the involvement of antigen presenting cells and reflects the effects of inhibition of FoxO1 in CD4 T cells.

The balance between T effector and T regulatory cells is critical for the normal immune function. A Teff:Treg balance skewed towards Teffs favors autoimmunity, while therapeutically restoring Teff:Treg balance may lead to resolution of inflammation and amelioration of autoimmunity. Our data suggest that the suppression of T cell encephalitogenicity by AS1842856 may involve both Teff and Treg populations via shifting Teff: Treg balance in a way benefiting the resolution of inflammation and ameliorating autoimmunity. Previous evidence have suggested that FoxO1 may serve as a predecessor at Foxp3-binding loci in precursor cells (Samstein et al., 2012) and is one of the highest-ranking nodes downstream of SGK1 (Wu et al., 2013). More detailed molecular analyses including ChIP-Seq and DNase-Seq will help to reveal the detailed molecule mechanisms, which may lead to a better understanding of the role of FoxO1 in regulating autoreactive T cells and CNS autoimmunity.

5. Conclusions

Our data illustrated an important role of FoxO1 signaling in regulating autoreactive CD4 cells in CNS autoimmunity via suppressing encephalitogenic Teff cells and shifting Teff: Treg balance.

Funding

This study was supported by grants from National MS society to Y Yang (PP-1708-29110) and NIH to Y Yang (1R01NS088437-01A1).

List of abbreviations

MS

Multiple Sclerosis

CNS

central nervous system

EAE

experimental autoimmune encephalomyelitis

Teff

T effector cells

FoxO1

Forkhead box O 1

MBP

myelin basic protein

PLP

proteolipid protein

MOG

myelin oligodendrocyte glycoprotein

PD-1

immune checkpoint programmed cell death protein-1

PBMCs

Peripheral blood mononuclear cells

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ethics approval and consent to participate

The protocols used for these experiments received prior approval by the OSU Institutional Animal Care and Use Committee and were conducted in accordance with the United States Public Health Service’s Policy on Humane Care and Use of Laboratory Animals. The study on human cells was performed under OSU Internal Review Board protocol # 2015H0076 with written informed consent received from participants prior to inclusion in the study.

Declaration of Competing Interest

The authors declare that they have no competing interests with the contents in this paper.

Data availability statement

The data from this manuscript are available from the corresponding author upon reasonable request.

References

  1. Aqel SI, Yang X, Kraus EE, Song J, Farinas MF, Zhao EY, et al. A STAT3 inhibitor ameliorates CNS autoimmunity by restoring Teff:Treg balance. JCI Insight. 2021;6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arasanz H, Gato-Canas M, Zuazo M, Ibanez-Vea M, Breckpot K, Kochan G, et al. PD1 signal transduction pathways in T cells. Oncotarget. 2017;8:51936–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arellano B, Graber DJ, Sentman CL. Regulatory T cell-based therapies for autoimmunity. Discov Med. 2016;22:73–80. [PMC free article] [PubMed] [Google Scholar]
  4. Battaglia M, Stabilini A, Migliavacca B, Horejs-Hoeck J, Kaupper T, Roncarolo MG. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. Journal of immunology. 2006;177:8338–47. [DOI] [PubMed] [Google Scholar]
  5. Boettler T, von Herrath M. IL-7 receptor alpha blockade, an off-switch for autoreactive T cells. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:12270–1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cabrera-Ortega AA, Feinberg D, Liang Y, Rossa C Jr., Graves DT. The Role of Forkhead Box 1 (FOXO1) in the Immune System: Dendritic Cells, T Cells, B Cells, and Hematopoietic Stem Cells. Critical reviews in immunology. 2017;37:1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter LL, Leach MW, Azoitei ML, Cui J, Pelker JW, Jussif J, et al. PD-1/PD-L1, but not PD-1/PD-L2, interactions regulate the severity of experimental autoimmune encephalomyelitis. Journal of neuroimmunology. 2007;182:124–34. [DOI] [PubMed] [Google Scholar]
  8. Das J, Ren G, Zhang L, Roberts AI, Zhao X, Bothwell AL, et al. Transforming growth factor beta is dispensable for the molecular orchestration of Th17 cell differentiation. The Journal of experimental medicine. 2009;206:2407–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deng Y, Wang F, Hughes T, Yu J. FOXOs in cancer immunity: Knowns and unknowns. Semin Cancer Biol. 2018;50:53–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Diep CH, Charles NJ, Gilks CB, Kalloger SE, Argenta PA, Lange CA. Progesterone receptors induce FOXO1-dependent senescence in ovarian cancer cells. Cell Cycle. 2013;12:1433–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dominguez-Villar M, Baecher-Allan CM, Hafler DA. Identification of T helper type 1-like, Foxp3+ regulatory T cells in human autoimmune disease. Nature medicine. 2011;17:673–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dong G, Wang Y, Xiao W, Pacios Pujado S, Xu F, Tian C, et al. FOXO1 regulates dendritic cell activity through ICAM-1 and CCR7. Journal of immunology. 2015;194:3745–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Francisco LM, Salinas VH, Brown KE, Vanguri VK, Freeman GJ, Kuchroo VK, et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. The Journal of experimental medicine. 2009;206:3015–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frohman EM, Racke MK, Raine CS. Multiple sclerosis--the plaque and its pathogenesis. The New England journal of medicine. 2006;354:942–55. [DOI] [PubMed] [Google Scholar]
  15. Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, et al. Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling. Nature. 2010;467:967–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goverman J, Woods A, Larson L, Weiner LP, Hood L, Zaller DM. Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell. 1993;72:551–60. [DOI] [PubMed] [Google Scholar]
  17. Guery L, Hugues S. Th17 Cell Plasticity and Functions in Cancer Immunity. Biomed Res Int. 2015;2015:314620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hedrick SM, Hess Michelini R, Doedens AL, Goldrath AW, Stone EL. FOXO transcription factors throughout T cell biology. Nature reviews Immunology. 2012;12:649–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hornsveld M, Dansen TB, Derksen PW, Burgering BMT. Re-evaluating the role of FOXOs in cancer. Semin Cancer Biol. 2018;50:90–100. [DOI] [PubMed] [Google Scholar]
  20. Juchem KW, Sacirbegovic F, Zhang C, Sharpe AH, Russell K, McNiff JM, et al. PD-L1 Prevents the Development of Autoimmune Heart Disease in Graft-versus-Host Disease. Journal of immunology. 2018;200:834–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kallies A, Good-Jacobson KL. Transcription Factor T-bet Orchestrates Lineage Development and Function in the Immune System. Trends in immunology. 2017;38:287–97. [DOI] [PubMed] [Google Scholar]
  22. Kamali AN, Noorbakhsh SM, Hamedifar H, Jadidi-Niaragh F, Yazdani R, Bautista JM, et al. A role for Th1-like Th17 cells in the pathogenesis of inflammatory and autoimmune disorders. Mol Immunol. 2019;105:107–15. [DOI] [PubMed] [Google Scholar]
  23. Karmaus PWF, Chen X, Lim SA, Herrada AA, Nguyen TM, Xu B, et al. Metabolic heterogeneity underlies reciprocal fates of TH17 cell stemness and plasticity. Nature. 2019;565:101–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kebir H, Ifergan I, Alvarez JI, Bernard M, Poirier J, Arbour N, et al. Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis. Annals of neurology. 2009;66:390–402. [DOI] [PubMed] [Google Scholar]
  25. Kerdiles YM, Beisner DR, Tinoco R, Dejean AS, Castrillon DH, DePinho RA, et al. Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor. Nature immunology. 2009;10:176–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kerdiles YM, Stone EL, Beisner DR, McGargill MA, Ch’en IL, Stockmann C, et al. Foxo transcription factors control regulatory T cell development and function. Immunity. 2010;33:890–904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kroner A, Schwab N, Ip CW, Ortler S, Gobel K, Nave KA, et al. Accelerated course of experimental autoimmune encephalomyelitis in PD-1-deficient central nervous system myelin mutants. The American journal of pathology. 2009;174:2290–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kurebayashi Y, Nagai S, Ikejiri A, Ohtani M, Ichiyama K, Baba Y, et al. PI3K-Akt-mTORC1-S6K1/2 axis controls Th17 differentiation by regulating Gfi1 expression and nuclear translocation of RORgamma. Cell reports. 2012;1:360–73. [DOI] [PubMed] [Google Scholar]
  29. Laine A, Martin B, Luka M, Mir L, Auffray C, Lucas B, et al. Foxo1 Is a T Cell-Intrinsic Inhibitor of the RORgammat-Th17 Program. Journal of immunology. 2015;195:1791–803. [DOI] [PubMed] [Google Scholar]
  30. Langlet F, Haeusler RA, Linden D, Ericson E, Norris T, Johansson A, et al. Selective Inhibition of FOXO1 Activator/Repressor Balance Modulates Hepatic Glucose Handling. Cell. 2017;171:824–35 e18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Latchman YE, Liang SC, Wu Y, Chernova T, Sobel RA, Klemm M, et al. PD-L1-deficient mice show that PD-L1 on T cells, antigen-presenting cells, and host tissues negatively regulates T cells. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:10691–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Laurence A, Amarnath S, Mariotti J, Kim YC, Foley J, Eckhaus M, et al. STAT3 transcription factor promotes instability of nTreg cells and limits generation of iTreg cells during acute murine graft-versus-host disease. Immunity. 2012;37:209–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lee LF, Logronio K, Tu GH, Zhai W, Ni I, Mei L, et al. Anti-IL-7 receptor-alpha reverses established type 1 diabetes in nonobese diabetic mice by modulating effector T-cell function. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:12674–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lovett-Racke AE, Yang Y, Racke MK. Th1 versus Th17: are T cell cytokines relevant in multiple sclerosis? Biochimica et biophysica acta. 2011;1812:246–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Luo CT, Liao W, Dadi S, Toure A, Li MO. Graded Foxo1 activity in Treg cells differentiates tumour immunity from spontaneous autoimmunity. Nature. 2016;529:532–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McFarland HF, Martin R. Multiple sclerosis: a complicated picture of autoimmunity. Nature immunology. 2007;8:913–9. [DOI] [PubMed] [Google Scholar]
  37. Nagashima T, Shigematsu N, Maruki R, Urano Y, Tanaka H, Shimaya A, et al. Discovery of novel forkhead box O1 inhibitors for treating type 2 diabetes: improvement of fasting glycemia in diabetic db/db mice. Mol Pharmacol. 2010;78:961–70. [DOI] [PubMed] [Google Scholar]
  38. Nistala K, Adams S, Cambrook H, Ursu S, Olivito B, de Jager W, et al. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:14751–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Onuma H, Vander Kooi BT, Boustead JN, Oeser JK, O’Brien RM. Correlation between FOXO1a (FKHR) and FOXO3a (FKHRL1) binding and the inhibition of basal glucose-6-phosphatase catalytic subunit gene transcription by insulin. Mol Endocrinol. 2006;20:2831–47. [DOI] [PubMed] [Google Scholar]
  40. Ouyang W, Beckett O, Ma Q, Paik JH, DePinho RA, Li MO. Foxo proteins cooperatively control the differentiation of Foxp3+ regulatory T cells. Nature immunology. 2010;11:618–27. [DOI] [PubMed] [Google Scholar]
  41. Penaranda C, Kuswanto W, Hofmann J, Kenefeck R, Narendran P, Walker LS, et al. IL-7 receptor blockade reverses autoimmune diabetes by promoting inhibition of effector/memory T cells. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:12668–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Polanczyk MJ, Hopke C, Vandenbark AA, Offner H. Treg suppressive activity involves estrogen-dependent expression of programmed death-1 (PD-1). International immunology. 2007;19:337–43. [DOI] [PubMed] [Google Scholar]
  43. Qu Y, Zhang B, Zhao L, Liu G, Ma H, Rao E, et al. The effect of immunosuppressive drug rapamycin on regulatory CD4+CD25+Foxp3+T cells in mice. Transpl Immunol. 2007;17:153–61. [DOI] [PubMed] [Google Scholar]
  44. Rao RR, Li Q, Gubbels Bupp MR, Shrikant PA. Transcription factor Foxo1 represses T-bet-mediated effector functions and promotes memory CD8(+) T cell differentiation. Immunity. 2012;36:374–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rautio J, Meanwell NA, Di L, Hageman MJ. The expanding role of prodrugs in contemporary drug design and development. Nat Rev Drug Discov. 2018;17:559–87. [DOI] [PubMed] [Google Scholar]
  46. Sakaguchi S, Vignali DA, Rudensky AY, Niec RE, Waldmann H. The plasticity and stability of regulatory T cells. Nature reviews Immunology. 2013;13:461–7. [DOI] [PubMed] [Google Scholar]
  47. Salama AD, Chitnis T, Imitola J, Ansari MJ, Akiba H, Tushima F, et al. Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. The Journal of experimental medicine. 2003;198:71–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Samstein RM, Arvey A, Josefowicz SZ, Peng X, Reynolds A, Sandstrom R, et al. Foxp3 exploits a preexistent enhancer lsandscape for regulatory T cell lineage specification. Cell. 2012;151:153–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Savai R, Al-Tamari HM, Sedding D, Kojonazarov B, Muecke C, Teske R, et al. Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension. Nature medicine. 2014;20:1289–300. [DOI] [PubMed] [Google Scholar]
  50. Tan CL, Kuchroo JR, Sage PT, Liang D, Francisco LM, Buck J, et al. PD-1 restraint of regulatory T cell suppressive activity is critical for immune tolerance. The Journal of experimental medicine. 2021;218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. van Langelaar J, van der Vuurst de Vries RM, Janssen M, Wierenga-Wolf AF, Spilt IM, Siepman TA, et al. T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention. Brain. 2018;141:1334–49. [DOI] [PubMed] [Google Scholar]
  52. Wallin MT, Culpepper WJ, Campbell JD, Nelson LM, Langer-Gould A, Marrie RA, et al. The prevalence of MS in the United States: A population-based estimate using health claims data. Neurology. 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wang F, Demir S, Gehringer F, Osswald CD, Seyfried F, Enzenmuller S, et al. Tight regulation of FOXO1 is essential for maintenance of B-cell precursor acute lymphoblastic leukemia. Blood. 2018;131:2929–42. [DOI] [PubMed] [Google Scholar]
  54. Wu C, Yosef N, Thalhamer T, Zhu C, Xiao S, Kishi Y, et al. Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1. Nature. 2013;496:513–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Xu J, Yuan S, Tian J, Martin KA, Song J, Li C, et al. Ultrasound mediated delivery of oxygen and LLL12 loaded stimuli responsive microdroplets for the treatment of hypoxic cancer cells. Sci Rep. 2017;7:44908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yang XO, Nurieva R, Martinez GJ, Kang HS, Chung Y, Pappu BP, et al. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs. Immunity. 2008;29:44–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yang Y, Weiner J, Liu Y, Smith AJ, Huss DJ, Winger R, et al. T-bet is essential for encephalitogenicity of both Th1 and Th17 cells. The Journal of experimental medicine. 2009;206:1549–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zou P, Liu L, Zheng L, Liu L, Stoneman RE, Cho A, et al. Targeting FoxO1 with AS1842856 suppresses adipogenesis. Cell Cycle. 2014;13:3759–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zuazo M, Gato-Canas M, Llorente N, Ibanez-Vea M, Arasanz H, Kochan G, et al. Molecular mechanisms of programmed cell death-1 dependent T cell suppression: relevance for immunotherapy. Ann Transl Med. 2017;5:385. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data from this manuscript are available from the corresponding author upon reasonable request.

RESOURCES