Characterization of the immunomodulation by the forkhead transcription factor
FOXO3
vorgelegt von M.Sc. Jelka Hartwig
von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades
Doctor rerum naturalium -Dr.rer.nat.-
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Roland Lauster 1. Gutachter: Prof. Dr. Jens Kurreck
2. Gutachterin: Prof. Dr. Carmen Scheibenbogen
Tag der wissenschaftlichen Aussprache: 25. Juni 2019
Table of Content
Abstract ... 1
Zusammenfassung ... 2
1 Introduction ... 3
1.1 Activation and regulation of the immune system ... 3
1.1.1 Adaptive immune system ... 3
1.1.2 Innate immune system ... 5
1.2 Cellular redox homeostasis in immune cells ... 8
1.3 FOXOs ... 11
1.3.1 Expression of FOXO transcription factors ... 11
1.3.2 Intracellular signaling of FOXOs ... 11
1.3.3 Role of FOXO3 in regulation of cellular activation and homeostasis ... 13
1.4 Immunomodulatory effects of FOXO3... 17
1.5 SNPs in the FOXO3 gene and their association with longevity and inflammation .... 18
1.6 Cardiovascular Disease and FOXO3 ... 19
1.7 Metformin and salicylate in the immune system ... 20
1.7.1 Metformin ... 20
1.7.2 Salicylate... 22
1.8 Aim of this study ... 22
2 Experimental procedures ... 24
2.1 Material ... 24
2.1.1 Ragents and chemicals ... 24
2.1.2 Antibodies and dyes ... 25
2.1.3 Cell culture ... 27
2.2 Methods ... 28
2.2.1 Isolation of mononuclear cells ... 28
2.2.2 Isolation of mouse splenocytes and peripheral blood cells ... 28
2.2.3 Detection of AMPK activation using Immunoblot ... 28
2.2.4 Flow cytometry (FC) analysis in human PBMCs ... 29
FOXO3 activation ... 29
Intracellular cytokine measurement ... 30
2.2.5 FC in mouse splenocytes ... 33
2.2.6 SNP genotyping using TaqMan probes ... 34
2.2.7 RNA isolation and FOXO3 target gene expression ... 36
2.2.8 Statistical analysis ... 36
3 Results ... 38
3.1 Characterization of immune cells from WT and Foxo3-/- mice showed enhanced frequencies of T and myeloid cells and diminished B cells ... 38
3.2 Foxo3 negatively regulates NK cell function ... 39
3.2.1 NK cells of Foxo3-/- mice are more activated... 39
3.2.2 NK cells of Foxo3-/- mice do not have alterations in apoptosis ex vivo ... 40
3.2.3 Foxo3-/- mice have more effector NK cells ... 41
3.2.4 NK cells of Foxo3-/- mice show increased cytotoxicity and IFN-γ production ... 42
3.2.5 FOXO3 gain-of-function immune SNP rs12212067 is associated with altered NK cell function in human ... 42
3.3 FOXO3 gain-of-function immune SNP rs12212067 has no influence on immune functions/parameters in a CFS cohort ... 45
3.4 FOXO3 gain-of-function SNPs and their influence on ROS/RNS level in human PBMCs ... 50
3.5 FOXO3 can be modulated by metformin ... 54
3.5.1 Metformin activates AMPK in human immune cells ... 54
3.5.2 Metformin activates FOXO3 in human immune cells in an AMPK dependent manner... 56
3.5.3 Metformin activates FOXO3 target genes ... 59
3.5.4 Metformin reduces PMA-induced ROS/RNS production ... 60
3.5.5 ROS/RNS reduction by metformin in CD11b+ cells is FOXO3 mediated ... 64
3.5.6 Metformin inhibits TNF-α and IFN-γ in T and NK cells ... 66
3.5.7 Metformin does not influence regulatory T cell function (FoxP3 and IL-10) in human immune cells ... 67
3.5.8 Metformin induces anti-inflammatory IL-10 in regulatory B cells ... 69
4.1 Enhanced NK cell activation due to Foxo3 deficiency in mice ... 72
4.2 FOXO3 gain-of-function SNPs have no influence on immune parameters and functions in human immune cells ... 73
4.3 FOXO3 can be modulated pharmacologically with the antidiabetic drug metformin in human immune cells ... 76
5 References ... 81
6 List of Abbreviation ... 95
7 Supplemental data ... 98
8 Acknowledgment ... 101
Jelka Hartwig Abstract
A
BSTRACTForkhead box O3 (FOXO3) is a transcription factor involved in cell metabolism, survival, longevity and inflammatory diseases. In the current thesis, we provided evidence that deletion of Foxo3 in mice enhances natural killer (NK) cell development, activity, cytotoxic function and IFNgamma (IFN-γ) expression leading to a better viral clearance in Coxsackievirus B3 (CVB3) infected hearts 3 days after infection and a lower inflammatory destruction of the heart tissue 7 days after infection. In line with this, we described an association of the gain-of-function single nucleotide polymorphism (SNP) rs12212067 with reduced IFN-γ production in NK cells. Metformin comprehensive pathway and functional analysis provided evidence that metformin activates FOXO3 via AMP-activated protein kinase (AMPK). Further, reactive oxygen and nitrogen species (ROS/RNS) production was reduced in human immune cells accompanied by an induction of gene expression of antioxidative enzymes superoxide dismutase 2 (SOD2) and cytochrome c (CYCS) being FOXO3 targets. This effect may be beneficial in chronic inflammation and atherosclerosis and may play a role in the improved morbidity and mortality of diabetes patients taking metformin. Ongoing trials (TAME, MILES) analyze the effect of metformin on morbidity and mortality in healthy older people. Based on our findings it would be very interesting to study the effect of metformin on FOXO3 activation in clinical trials and the potential influence of FOXO3 SNPs.
Jelka Hartwig Zusammenfassung
Z
USAMMENFASSUNGDer Transkriptionsfaktor Forkhead-Box-Protein O3 (FOXO3) ist essentiell für die Regulation zahlreiche zelluläre Prozesse wie dem Metabolismus, der Proliferation und der Apoptose. Des Weiteren ist FOXO3 mit Langlebigkeit assoziiert und spielt eine Rolle bei der Immunzellregulation bei entzündlichen Erkrankungen.
Die Ergebnisse dieser Arbeit zeigten, dass der Verlust von Foxo3 in der Maus die Entwicklung natürliche Killerzellen (NK Zellen) fördert und deren Aktivität, Zytotoxizität und Interferon-gamma (IFN-γ) Expression verstärkt. Dies führte zu einer verbesserten viralen Eliminierung in Coxsackievirus B3 (CVB3) infizierten Herzen und folglich zu einer geringeren entzündlich bedingten Schädigung des Herzgewebes. In humanen NK Zellen zeigte sich zudem, dass die expressionssteigernde Variante des Einzelnukleotid-Polymorphismus (SNP) rs12212067 im FOXO3 Gen mit einer geringeren IFN-γ Produktion assoziiert ist.
Umfangreiche Untersuchungen von induzierten Signalwegen und Funktionen in humanen Immunzellen zeigten weiter, dass das Antidiabetikum Metformin FOXO3 abhängig von der AMP-aktivierte Proteinkinase (AMPK) aktiviert. Die FOXO3 Aktivierung führte weiter zur Induktion antioxidativ wirkender FOXO3 Zielgene: der mitochondrialen Superoxide dismutase 2 (SOD2) und des Cytochrom c (CYCS) und folglich zu einer Abnahme reaktiver Sauerstoff- und Stickstoffspezies (ROS/RNS). Eine Aktivierung von FOXO3 durch Metformin in Immunzellen kann bei chronischen Entzündungen sowie Atherosklerose vorteilhaft sein und auch die Verbesserung der Morbidität sowie Mortalität bei Patienten mit Diabetes unter Metformin-Therapie erklären. Laufende Studien (TAME, MILES) untersuchen zurzeit die Wirkung von Metformin auf die Morbidität und Mortalität bei älteren Menschen. Auf Grundlage der vorliegenden Arbeit wäre hierbei eine erweiterte Untersuchung der Bedeutung von FOXO3 Aktivierung unter Metformin-Therapie und der potentielle Einfluss von FOXO3 SNPs von weitreichendem Interesse.
Jelka Hartwig Introduction
1 I
NTRODUCTIONThe transcription factor Forkhead box O3 (FOXO3), a member of the forkhead box O family, plays critical roles in diverse cellular functions including proliferation, apoptosis, and metabolism Eijkelenboom and Burgering (1), (2). In immune cells, FOXO3 activity is critical for maintenance of immune progenitor cell homeostasis (3-5) and its deficiency in B and T cells leads to their enhanced activity during infection (6). A role of FOXO3 as a powerful immune break was discussed in several studies (7, 8). In patients with inflammatory diseases such as rheumatoid arthritis and IBD, genome-wide association studies have shown that individuals with a single nucleotide polymorphism (SNP) in the FOXO3 gene locus leading to lowered FOXO3 expression have a more aggressive disease course (9, 10).
1.1 Activation and regulation of the immune system
1.1.1 Adaptive immune systemThe adaptive immune system can be divided into a central and a peripheral one. Central lymphoid organs are bone marrow and thymus that gives rise to T and B cells. These cells than can migrate via the blood system to the peripheral lymphoid organs like lymph nodes, spleen, tonsils and other mucosal associated lymphoid tissues. The adaptive immune system contains B and T cells, that have formed highly specific receptors and that are able to form an immunological memory. Central for the activation of T cells are dendritic cells (DCs) via major histocompatibility complex (MHC)/ T cell receptor (TCR) interaction (11). If an antigen appeared a second time, the immune reaction can evolve rapidly. Antibodies, secreted by B cells bind pathogens which in term leads to its neutralization, opsonisation and complement activation as a part of the humoral immune system (12-14).
T cells are highly specialized lymphocytes and account for 70% of the lymphocytes in human blood. They originate from the bone marrow, develop in the thymus and are responsible for the regulation of immune responses, specific elimination of pathogens and play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes like B and NK cells by bearing a TCR which enables the specific recognition of pathogens, infected and transformed cells. To fulfill these functions T cells pass various steps of selection preventing
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peripheral lymphoid organs. The TCR is associated with the cluster of differentiation (CD) 3 protein complex. The CD3 molecule is responsible for the signal transduction of the TCR and its translocation to the T cell surface. Other co-receptors associated with the TCR are CD4 and CD8 molecules, dividing them into the major classes: CD4+ T helper cells and CD8+ cytotoxic T cells. These co-receptors mediate the interaction of the TCR with the constant region of the MHC class I or II molecules during the antigen recognition of T cells. T cells are activated in peripheral lymphoid organs by APCs presenting processed antigens via MHC molecules. CD8+ cytotoxic T cells protect the body by killing cells which are transformed and infected with intracellular pathogens. These cells recognize their targets by binding to MHC class I molecules loaded with peptide antigens. Most effector function of CD8+ T cells depend on the presence of interleukin (IL)-2 and IFNgamma (IFN-γ) released by CD4+ T cells. CD4+ helper cells activate and direct other immune cells. They are essential for the antibody production by B cells, activate and help CD8+ cytotoxic T cells and maximize the effector function of macrophages. Activation of CD4+ cells by loaded MHC class II molecules and costimulatory signals induce the release of cytokines and the differentiation into subclass of CD4+ T cells, among them T helper (Th) 1 or Th2 cells. Th1 cells produce IFN-γ, IL-2 and TNF-α and promote macrophages to kill phagocytosed pathogens. Th2 cells secrete IL-4, IL-5 IL-6, IL-10 and IL-13 and stimulate the differentiation of B cells into plasma cells as well as the production of antibodies (14, 15).
B cells, express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a specific antigen, against which it will initiate an antibody response (14). B cells also belong to the lymphocyte lineage and develop from CD34+ stem cells in the bone marrow. The development from immature B cells is accompanied by V (D)J-recombination of the B cell receptor (BCR), leading to a high specificity of the receptor. After several selection processes B cells migrate out of the bone marrow as transitional B cell via the peripheral blood into secondary lymphoid organs like spleen and lymph nodes. There B cells develop into follicular naïve B cells and marginalzone-like memory B cells. This process is T cell independent. With T cell dependent antigen contact activation of naïve B cells occurs. Now the B cells starts to expand clonally (16). Via affinity maturation together with somatic hypermutation and class-switch recombination, which is the exchange of the heavy chain, different high affinity antibodies are developed (IgG, IgE, IgA). Isotype switched memory B cells and plasmablasts are
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generated. The latter migrate back into the bone marrow were they persist as long-lived plasmablast able to secrete antibodies within short notice (13, 14, 17).
Once activated the immune system clears the pathogenic invasion by various mechanisms described in this chapter. To resolve an immune response different regulatory cell types exist including regulatory CD4+ T cells (Tregs) (18-21) and regulatory B cells (Bregs) (22, 23), regulatory γδ cells (24), regulatory CD8+ T cells (CD8+ Tregs)(25), and regulatory innate lymphoid cells (ILCregs) (26, 27). Treg cell–mediated immune regulation is mediated by high IL-2R expression on Treg cells that could deprive effector T cells of IL-2 and thereby inhibit their proliferation (28). Also CTLA-4, mediates Treg cell–mediated suppressor function by inhibiting the activation of T cells by DCs (29). In addition secreted proteins, including IL-10, IL-35, granzyme B, IL-9 and TGF-β have been implicated in Treg mediated suppression (reviewed in (30, 31). Selective ablation of IL-10 in FoxP3+ Treg cells revealed that IL-10 production by Treg cells is essential for keeping the immune response in check at environmental interfaces such as colon and lung (32). Furthermore, TGF-β produced by Treg cells can suppress Th1 responses (33) and upon activation, Treg cells can kill either responder T cells or APCs in a granzyme B–dependent manner in vitro (34, 35). The mechanisms of Breg-mediated immunosuppression include cell contact-mediated mechanisms and anti-inflammatory cytokine secretion (e.g. IL-10, TGF-β) (36). Bregs suppress CD4+ T-cell proliferation, Th1/17 differentiation, and CD8+ effector T-cell activity. They induce the expansion of FoxP3+ Tregs. and conversion of naïve CD4+ T cells into FoxP3+ Tregs, which is mediated by TGF-β secretion and cell-to-cell contact between T and B cells (37), which may contribute to the global suppression of inflammatory responses. Furthermore, Bregs release IL-10 after recognition of activated CD4+ T cells that express CD40L and induce their anergy (38).
1.1.2 Innate immune system
Invading pathogens like bacteria and viruses are first recognized by cells of the innate immune system like NK cells, monocytes, macrophages and neutrophils. These cells have receptors called Toll-like receptors (TLRs) that recognize different pathogen-associated molecular pattern like, DNA, RNA, and cell membrane components which are broadly shared by pathogens (12-14).
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Monocytes are the largest type of leukocyte and can differentiate into macrophages and myeloid lineage DCs. Monocytes have a bean-shaped nucleus and a granulated cytoplasm (39). Monocytes are produced by the bone marrow from precursors called monoblasts. They circulate in the bloodstream for one to three days. About half of the body's monocytes are stored as a reserve in the spleen (40). Monocytes compose 2-10% of leukocytes in the human body and serve multiple roles in immune function including restock resident macrophages under normal conditions. There are at least three types of monocytes in human blood: classical monocytes (CD14++,CD16−monocyte), non-classical monocyte (CD14+CD16++monocyte) and intermediate monocyte (CD14++CD16+monocytes) (41, 42). They migrate within approximately 8–12 h in response to inflammation signals from infected tissues and differentiation into macrophages or DCs to trigger an immune response (39, 40). Monocytes and their progeny (macrophages, DCs) serve three main functions in the immune system. These are phagocytosis, antigen presentation, and cytokine production. Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Many factors produced by other cells can regulate the chemotaxis and functions of monocytes. These factors include most particularly chemokines such as monocyte chemotactic protein-1 (CCL2) and monocyte chemotactic protein-3 (CCL7) (43). Microbial fragments can serve as antigens when incorporated into MHC molecules. This process is called antigen presentation and it leads to activation of T cells. Other microbial products can directly activate monocytes, and this leads to production of pro-inflammatory and delayed of anti-inflammatory cytokines. Typical cytokines produced by monocytes are TNF-α, IL-1, and IL-12 (14).
NK cells are key players and the first line of defense of the innate immune system and are involved in the regulation of the immune response. NK cells mediate cytotoxicity against tumour and virus infected cells. IFN-γ production displays the important defense mechanism of NK cells which are regulated by a wide range of receptors, cytokines and hormones. Consistent with data in mouse the human NK cell turnover in blood is around two weeks (44, 45). Human NK cells comprise 2-18% of total lymphocytes in peripheral blood and spleen whereas murine NK cells represent around 2% of splenocytes (46). The mechanism by which NK cells recognize and discriminate target cells from healthy cells depends on a dynamic equilibrium of inhibitory and activation receptor signaling through corresponding NK cell receptors. Inhibitory NK cell receptors detect the presence of self-molecules, MHC class I
Jelka Hartwig Introduction
molecules on potential target cells through MHC class I-specific receptors. The absence of MHC class I molecules on target cells, downregulated by viruses, in transformed cells and upon stress, results in a loss of the inhibitory signal and leads to NK cell mediated cytotoxicity (47) via induction of apoptosis. Activating receptors on NK cells, comprise NKG2D and NKp46 recognizing ligands on target cells expressed upon stress and Toll-like receptors binding infectious non-self-ligands. Bearing several TLRs, in vitro exposure of NK cells to TLR ligands (TLR-L) such as lipopolysaccharide (LPS) results in enhanced IFN-γ production and increased cytotoxicity (48).
Distinct NK cell subsets have been defined and characterized in human as well as in mice based on phenotypic, functional and anatomic features. Human NK cells are identified by the absence of the surface molecule CD3 and the expression of CD56 (neural cell adhesion molecule). These lymphocytes further can be divided into CD56dim and CD56bright subsets differing in their function and phenotypic properties. 90% of peripheral NK cell are CD56dim. CD56dim NK cells display enhanced cytotoxicity via perforin and granzyme than the CD56bright subset. CD56bright NK cells produce large amounts of cytokines such as IFN-γ in response to stimulation with TLR-L and ITLR-Ls (49). Consequently, CD56bright and CD56dim NK cells have been described as two functionally distinct subsets, cytokine producing and cytolytic, respectively. However, several observations have challenged this strict separation, as both subsets can be cytotoxic or produce cytokines, after appropriate in vitro stimulation. Upon target cell recognition, resting CD56dim NK cells are highly cytotoxic, but can produce cytokines as well (50-52). In contrast, CD56bright NK cells require cytokine activation (combinations of IL2, IL12, IL15, IL18) to induce cytotoxic activity and produce cytokines (50, 51, 53).Both subsets express a high affinity IL-2 receptor and are therefore able to proliferate in response to low dose of IL-2. Furthermore, constitutively receptors for monocyte-derived cytokines such as 1, 10, 12, 15 and IL-18 are expressed on both subtypes. Thus their function and activation can be mediated by monocytes (49).Several groups proposed that CD56bright cells are precursors of the CD56dim NK subset displaying longer telomere length and the ability to differentiate in contact with fibroblasts into CD56dim NK cells (54, 55). Mouse NK cells mainly characterized by the expression of NKp46 and DX5 and the absence of CD3 can be subdivided into four subsets, based on their developmental expression of CD11b (Mac-1) and CD27 (56). The maturation process starts at
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and CD11bhighCD27low considered to be the most mature NK cell type (56-58). The CD11blow subset is mostly found in bone marrow, lymph nodes and liver whereas CD11bhigh NK cells display the majority in spleen, peripheral blood and lung. Further the CD11blow subset can be divided into CD27high and CD27low NK cells. Both subsets, CD11blowCD27low and CD11blowCD27high, are closely related to each other but differ in their proliferative potential with CD27low having the highest potential to proliferate and giving rise to all other subsets. Hence, the CD11blowCD27low NK cells are suggested to be the immature NK cells. The CD11bhigh subset can also be further subdivided into CD27low and CD27high NK cells with the CD11bhighCD27high subset displaying the most potent effector cells. Moreover, the CD11bhighCD27high subset produce higher amounts of IFN-γ compared to CD11bhighCD27low NK cells and possesses a lower threshold to be activated (58).
1.2 Cellular redox homeostasis in immune cells
Reactive oxygen species (ROS) are highly reactive oxygen containing molecules. They are either produced exogenously or endogenously (59). Concentrations of ROS and reactive nitrogen species (RNS) seem to determine the cell fate. Hence, high ROS/RNS level cause damage on lipids, proteins and DNA, whereas low concentrations seem to have important roles in immunomodulation and signaling (60, 61).
The main endogenous cell sources of reactive species are the mitochondria, in which mitochondrial (m)ROS is generated as a byproduct of ATP producing cellular respiration. Another source is the membrane bound NADPH-dependent oxidases (NOX), which are transmembrane flavocytochrome proteins. They generate ROS by reducing molecular oxygen to superoxide anion (O2-) and hydrogen peroxide (H2O2). This production of ROS is used by activated neutrophils as a weapon against invading pathogens known as “respiratory burst” (59, 62). Further endogenous sources of ROS/RNS production comprise cytochrome p450 enzymes, lipoxygenases or nitric oxide synthases (NOs), latter generate NO also as a defense mechanism against pathogens (63). Exogenous sources of oxidants are, for instance, ozone exposure, ionizing radiation, heavy metal ions or drugs (61). ROS including free radicals (O2- and hydroxyl radicals), oxidants (H2O2) and electrophiles are involved in diseases such as cancer, cardiovascular complications, acute and chronic inflammation, and neurodegenerative diseases (64).
Jelka Hartwig Introduction
Maintenance of a cellular redox homeostasis is crucial for the adequate function of cellular processes including physiological immune mechanisms. Imbalances in the production and removal of ROS/RNS cause pathophysiological dysregulation in the immune response, being involved in chronic immune activation like infections, allergies, autoimmune or neurodegenerative diseases (65). The shift in balance in favor of oxidants is termed as “oxidative stress” (Figure 1) (66).
Figure 1: Regulation and disturbance of redox homeostasis. Under physiological conditions a balance exist between oxidants and antioxidants. Increase in ROS/RNS due to exogenous and/or endogenous factors induces an imbalance of the redox homeostasis termed oxidative stress. This situation is accompanied with oxidative damage of proteins, lipids and DNA. Modified from (67).
But the cell is not defenseless against those damaging ROS/RNS. Oxidative stress leads to activation of a battery of defensive gene expression that leads to detoxification of chemicals as well as ROS/RNS and prevention of free radical generation promoting cell survival. Different types of antioxidative molecules exist. Some are transcription factors associated with the cellular response to oxidative stress. For example, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heat shock factor 1 (HSF1), and nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) (68). The Nrf2 pathway is regarded as the most important in the cell to protect against oxidative stress (69). Nrf2 binds to Antioxidant response element (ARE) and regulates ARE-mediated gene expression, coordinating the induction of several antioxidant enzymes, like heme oxygenase 1, glutathione reduktase, glutathione S-transferase and NAD(P)H:quinone oxidoreduktasef (69-71). Besides the Nrf transcription factors activator complex 1 is involved
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in regulation of antioxidant genes via binding to 12-O-Tetradecanoylphorbol-13-acetate response element with the consensus sequence 5’-TGACTCA-3’ (72, 73).
Some enzymatic antioxidants for example thioredoxins (Trx), catalase or superoxide dismutase (SOD) catalyze the clearance of ROS/RNS within different subcellular compartments. glutathione (GSH) is one of the important endogenous antioxidants. It can either work directly or can work as a cofactor for other detoxifying enzymes (74). Additionally, when secreted decay products of GSH fill up extracellular buffer pool and the Trx when secreted has also chemokine-like properties attracting monocytes, neutrophils, and T cells (65). Besides GSH there are other non-enzymatic antioxidants like vitamin E, ascorbate, pyruvate or carotinoids (66).
For a long time ROS/RNS were considered as harmful. Now, there is strong evidence that ROS/RNS are important intra- and extracellular messengers controlling signaling pathways (59).
An important defense mechanism against invading microorganism is the “respiratory burst” of neutrophils and macrophages. During respiratory burst, the phagolysosome is filled with O2 -built by the NOX2 complex, which displays the main source of antimicrobial ROS (62, 75). O2 -can be converted into various other ROS like peroxynitrite (ONOO-), hypocholoric acid (HOCI) or H2O2. To circumvent massive tissue destruction and damage this process is under tight control and regulation (59). Neutrophil-derived ROS/RNS can also influence the immune response by activating pro-inflammatory signaling pathways like NF-κB and mitogen-activated protein kinases (MAPK) pathway (75).
But ROS/RNS do not only kill pathogens. Studies suggested that nod-like receptor pyrin domain-containing 3 inflammasome activity is negatively regulated by autophagy and positively regulated by ROS/RNS generated by mitochondria (60, 76). The inflammasome is part of the first line defense mechanisms of the innate immune system and induces the secretion of pro-inflammatory cytokines (76).
ROS, especially mROS derived from mitochondria, can also trigger the differentiation of DCs from monocyte precursors through a NF-κB dependent mechanism (59). A further task is the regulation of T cell proliferation, function and apoptosis (77) as well as the regulation of B cell activation and BCR signaling (78, 79).
Under metabolic stress like glucose deprivation subsequent ATP depletion and ROS accumulation activates AMP-activated protein kinase (AMPK). AMPK is a heterotrimer complex,
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including a catalytic subunit (α) and two regulatory subunits (β and γ). ROS directly activate AMPK through S-glutathionylation of cysteines on the AMPKα and β subunit (80). In addition, phosphorylation on the Threonine 172 (T172) in the presence of high AMP/ATP ratios due to ATP depletion enables it to act as an energy sensor for the cell (67, 81).
1.3 FOXOs
1.3.1 Expression of FOXO transcription factors
The FOXO family of transcription factors is the mammalian orthologue of Dauer Abnormal Formation-16 (DAF-16) in Caenorhabditis elegans (82). They have a highly conserved helix-loop-helix DNA binding domain (83) which enables the transcription factor to bind the same consensus sequence 5´-TTGTTTAC-3´ (1). In mammals four transcription factors belong to the family of FOXO. FOXO1 (also known as FKHR), FOXO3 (also known as FKHRL1), FOXO4 (also known as AFX1) and FOXO6 (2). Studies from mice reveal that Foxo1, Foxo3 and Foxo4 are expressed more ubiquitously (84), Foxo6 however is expressed mainly in brain tissue (85). Foxo1 expression was shown in B cells, T cells and ovaries, and deletion leads to embryonic lethality in mice due to abnormal angiogenesis (86, 87). Foxo3 is highly expressed in lymphocytes and myeloid cells. Deletion in mice cause only a mild phenotype with suppressed ovarian follicular activity and a spontaneous, multisystemic inflammatory syndrome associated with lymphadenopathy, increased NF-kB activation, and hyperactivation of T cells (87, 88).
1.3.2 Intracellular signaling of FOXOs
In mammals FOXOs are regulated by growth factors, oxidative stress or nutrient deprivation (1). Activity of FOXOs is regulated by transcription and via posttranslational modifications (ptm) such as phosphorylation, acetylation, methylation and ubiquitination (2). Those ptms influence the subcellular localization and orchestrate its shuttling in or from the nucleus were FOXOs fulfil their task (89).
AKT (also known as protein kinase B) and AMPK are the major antagonistic regulators of FOXO3 (Figure 2). The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-AKT and the liver kinase B1 (LKB1)-AMPK pathways integrate information about the nutrient availability in the cell. AMPK acts as a sensor of cellular energy charge adenosine triphosphate/ adenosine
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promotes longevity (90, 91). The PI3K-AKT pathway controls cell survival and cell proliferation but also tumor growth and aging (92-94). FOXO3 and mTOR (target of rapamycin) are the two major downstream effectors regulated by AMPK and AKT with antagonistic effects (67, 95). FOXO3 activation increases defense to oxidative stress by targeting the expression of antioxidative enzymes e.g. SOD, catalase and sestrin (96). In addition, FOXOs participate in glucose metabolism by regulating phosphoenolpyruvate carboxykinase (PEPCK), the key enzyme in gluconeogenesis and glucose-6-phosphatase (G6P) (97). In addition, FOXO induces expression of autophagy-related genes (ATG4, ATG6, ATG7, ATG12, etc.) (98).
mTOR, a protein kinase that plays a critical role in protein synthesis and cellular growth is activated by AKT and inhibited by AMPK (99, 100). Thus, the PI3K-AKT and the LKB1-AMPK pathways may orchestrate a series of FOXO3- and mTOR-mediated changes that allow the organism to adapt to alterations in nutrient status and oxidative stress (67). Under nutrient deprivation, AMPK inhibits mTOR activity, thereby decreasing protein synthesis and increasing autophagy. This reduced anabolism reduces ROS production, while enhanced autophagy and glycolysis increases the resistance of cells to ROS (67).
Oxidative stress or a decrease in cellular energy status triggers phosphorylation of FOXO3 by AMPK on six consensus sites (Thr179, Ser399, Ser413, Ser555, Ser588, and Ser626) leading to its activation and differential target gene expression (95, 101). AMPK can indirectly enhance FOXO3-mediated transcriptional activity by recruiting CREB-binding protein and p300 (1). An influence on FOXO3 localization by AMPK is controversially discussed. Greer et al. showed that reduced AMPK-dependent phosphorylation results only decreased FOXO3 activity but has no effect on the localization (95). Other studies suggest that AMPK facilitate FOXO3 nuclear localization (102). In addition, AMPK can increase nuclear FOXO acetylation, thereby mediating localization to nuclear promyelocytic leukemia bodies, which act as transcriptional co-activators (103). Phosphorylation by AKT causes nuclear exclusion and inactivation of FOXO3 (104). AKT- mediated phosphorylation results in increased binding of FOXO3 to the regulator 14-3-3 and cytoplasmic shuttling of both (1).Besides, AKT also promotes the ubiquitination of FOXO leading to its cytosolic degradation (105).
Taken together the current opinion is that AKT-site dephosphorylation leads to nuclear localization and an additional AMPK-site phosphorylation (low energy status) to an increase in
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FOXO3 transcriptional activation and/or formation and recruitment of a coactivator complex at the promotor of interest (95). This is presumably orchestrated by a “molecular code” that can convert external stimuli into selective cellular responses due to the distinct ptm pattern (95). Although FOXO1 and FOXO3 are structurally similar, AMPK phosphorylates FOXO3 more efficient than FOXO1 (95).
Figure 2: Intracellular signaling and regulation of FOXO3. Signaling of AMPK and AKT on ROS homeostasis via mTOR and FOXO regulation: Under metabolic stress, AMPK phosphorylates and activates FOXO3 thereby enhancing its transcriptional activity of glucose metabolic pathway genes (PEPCK, G6P) and antioxidant genes like SOD2. Simultaneously, AMPK inhibits mTOR and thereby the energy demanding protein synthesis pathway. AKT activates mTOR reversely and phosphorylates FOXO3 leading to the translocation from the nucleus to the cytoplasm. By ubiquitination of FOXO3, AKT leads to its degradation in cytoplasm. Modified from (67).
1.3.3 Role of FOXO3 in regulation of cellular activation and homeostasis
FOXOs main function is to orchestrate multiple information from upstream signaling mechanisms to enable tissue homeostasis during stress (1). It can directly modulate
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were first described as tumor suppressor genes, but several studies have shown that Foxo1 and Foxo3 play a great role in physiological and pathological immune responses (107-109). Nowadays it is clear that they are involved in many cellular processes regulating gene expression programs including cell cycle regulation, apoptosis, oxidative stress, metabolism, immunity and stem cell homeostasis in response to changes in nutrients, growth factors and also stress signals (Figure 3)(1, 2). The best analyzed role of FOXO3 is the regulation of insulin and insulin-like growth factor-1 receptor signaling important in stress response and longevity (82, 110). Genetic manipulations of FOXO3 lead to lifespan extension via an increased gene expression in various model organisms (92, 111). Enhanced FOXO3 transcription can mediate similar beneficial effects in humans (101, 112). In addition, FOXO3 activity can be modulated by dietary restriction (95, 113)and phytochemicals in green tea (114).
Hematopoietic stem cells (HSC)
FOXOs are essential for stem cell functions in diverse tissues, including also a central role in the maintenance of hematopoietic stem cell pool (115, 116) and homeostatic hematopoiesis (117). Mice studies showed that loss of Foxo3 alone significantly comprises bone marrow hematopoietic stem cell frequencies and regenerative properties leading to myeloproliferation (118), anemia (119) and immune deficiencies (120). The mechanisms by which FOXO3 regulates HSC quiescence are not fully elucidated. FOXO3 regulates some of the key genes involved in stem cell quiescence such as cyclin-dependent kinase (CDK) inhibitor 1B, cyclin G2, and p57 (121, 122). Furthermore, balancing ROS levels in HSC requires FOXO3 influencing DNA damage and thereby HSC maintenance and aging (123, 124).
Immune System
As described in more detail in section 1.3. FOXO3 is the most abundant isoform of the FOXO family in the myeloid compartment. It has recently been shown that it is a suppressor of inflammatory cytokines by DCs (120) and monocytes (9). Lee et al. showed that a SNP in the Foxo3 gene is associated with reduced pro-inflammatory and increased anti-inflammatory cytokine induction (9). This leads to a better prognosis in chronic inflammatory disorders like Crohn´s disease and rheumatoid arthritis but a poorer outcome in acute infection like malaria (9) and active tuberculosis (125). On the other hand, individuals infected with Trypanosoma cruzi causing chronic Chagasic cardiomyopathy showed no evidence of an association of FOXO3
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genetic variant rs12212067 with acute or chronic infection phase (126). Another important immune regulatory mechanism was discovered by Becker et al. 2010. They discovered that antimicrobial peptide expression was dependent on FOXO3 activity in vivo in Drosophila (127). Further studies showed that especially FOXO3 induces the synthesis of antimicrobial peptides in human bronchial epithelial cells (128).
Oxidative stress response
FOXO3 activation increases resistance to oxidative stress by targeting the expression of antioxidative enzymes like mitochondrial SOD2, catalase, sestrin, cytochrome c (CYCS1) and heme oxygenase 1 (HMOX1) (96)(see 1.2). Oxidative stress leads to induced nuclear localization of FOXO3 with increased expression of antioxidative, antiapoptotic, autophagy-related target genes in neonatal rat cardiomyocytes (129). It is furthermore essential for controlling ROS levels in HSC and thereby for the maintenance of the HSC pool (124, 130). Oxidative stress induced Jun N-terminalkinase (JNK) activates FOXO3. JNK induces the release of FOXOs from 14-3-3 regulator protein, thereby promoting FOXO3 nuclear localization (108). Oxidative stress can also effect sirtuin 1 (SIRT1) mediated deacetylation of FOXO3 leading to its activation (131). Metabolism
In addition, FOXO participates in metabolic pathways like the glucose metabolism by regulating PEPCK, G6P and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). PEPCK is the key enzyme in gluconeogenesis which is enhanced in energy deprivation and promotes glucose and glutamine metabolism (97, 132).
Others
Beside the ubiquitin-proteasome system, autophagy is a tightly regulated process involving the removal of damaged proteins and cell organelles mediated by lysosomal degradation and thereby is essential for maintenance of redox homeostasis (Yan et al. 2016). The first links between redox homeostasis and autophagy came from the investigation that starvation induces autophagic flux. Autophagy related genes are often activated by nutrient deprivation with the mTOR kinase acting as a critical regulator. Both PI3K/AKT and MAPK/Erk1/2 pathway activate mTOR which subsequently suppress autophagy, while AMPK and p53 signaling inhibit mTOR thereby promoting autophagy (133). Further analysis revealed that one molecular target
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induces transcription of autophagy-related genes, including ATG4, MAP-LC3 and Rab7 (98). Furthermore, FOXO3 acts as a cell surveillance mediator. If autophagy is malfunctioning FOXO3 links autophagy to apoptosis by inducing the pro-apoptotic gene BBC3/PUMA expression (134). Becker et al. showed that Foxo3 upregulates the pro-apoptotic members p53 up-regulated modulator of apoptosis (Puma) and Bcl-2-like protein 11 (Bim), leading to T cell apoptosis (127). Furthermore, following cytokine or growth factor withdrawal, FOXO3 promotes cell growth inhibitory signaling pathways by inducing multiple pro-apoptotic members of the B-cell lymphoma 2 (Bcl2) family of mitochondrial targeting proteins and enhancing the expression of various CDK inhibitors (135). One possible mechanism by which FOXO3 gets activated by SIRT1 dependent acetylation was shown by Brunet et al. who stated that SIRT1 has a dual effect on FOXO3. It promotes FOXO3s ability to induce cell cycle arrest and resistance to oxidative stress but it inhibits the FOXO3 induced cell death (131).
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Figure 3: Cellular processes regulated by FOXO3 that preserve cellular homeostasis in response to stress. FOXO3 protects against several diseases and induces healthy aging and lifespan extension. Modified from (101).
1.4 Immunomodulatory effects of FOXO3
Although FOXO proteins are expressed in immune cells, their physiological role in immune responses is not fully understood. The immunomodulatory function of Foxo3 in the adaptive immune response hast been elucidated to some extent. As Foxo3 is critical for lymphoid homeostasis by modulating NF-κB activity (6, 136), mice lacking Foxo3 showed a phenotype of lymphadenopathy, T Cell hyperproliferation, and autoreactivity (6). During antigen receptor stimulation of T and B cells, PI3K gets activated that consequently down regulates Foxo3 via Akt thus inducing proliferation (137, 138).
Its anti-inflammatory actions involve inhibition of production of inflammatory cytokines such as IL-2 (6) and IL-6 (120) influencing the production of antibodies, the function of T cells,
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Intensive studies have also revealed that Foxo3 is a key suppressor of inflammatory cytokine production by DCs and macrophages (120). By alterations in DC cytokine expression (IL-6) it also controls the magnitude of T cell immune response (120). A more recent study by Stienne et al. also identified Eomes as a direct target gene for Foxo3 in CD4+ T cells, having a specific role in the polarization of CD4+ T cells toward pathogenic Th1 cells producing IFN-γ and granulocyte monocyte colony stimulating factor (GM-CSF) (141). Within the adaptive immune system it has been shown that Foxo1 and Foxo3 cooperatively control the development and function of FoxP3+ Treg cells in mice (142, 143). Furthermore, Foxo3 limits expansion of memory CD8+ T cells during acute or chronic viral infection (144) and prevents CD4+ central memory cells from apoptosis (145). Inactivation of FOXO3 by IL-2 plays also a critical role in T cell proliferation and survival due to downregulation of CDK inhibitor p27Kip1 and pro-apoptotic Bim (146). The role of FOXO3 on cytokine production has already been elucidated to some extent. FOXO3 for example is able to antagonize signaling intermediates downstream of TLR4, such as NF-κB and Interferon regulatory factors (IRFs), resulting in inhibition of IFN-ß (147). But FOXO3 does not influence cytokine production (IFN-γ and TNF-α) in CD8+ cells (148).
1.5 SNPs in the FOXO3 gene and their association with longevity and
inflammation
Human longevity is a complex phenotype and the identification of the involved genes and variants is still challenging. Already 1994 apolipoprotein E allele ε4 was reported as the first genetic association with longevity(149). In the meantime variations in the FOXO3 gene were identified to influence longevity across diverse populations, including Japanese-Americans (150), Germans (151), and Danes (152). Flachsbart et al. showed that the effect of the FOXO3 association is very strong in long-lived individuals beyond 95 years of age, and especially in centenarians (151). Although FOXO3 is nowadays consistently described as a human longevity gene, the functional variants and the underlying mechanisms have not been identified yet. Most of the SNPs are located in the 3′- UTR of the gene and cluster in or near the last intron (151).It is believed that the important variants involved in healthy aging are not coding (153, 154). Genetic manipulations of FOXO3 orthologues lead to lifespan extension in various model organisms, mainly via an increased expression of the gene (111, 155). Enhanced FOXO3 transcription may also mediate a similar beneficial effect in humans (101). Flachsbart et al
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identified and characterized functional variants in the FOXO3 gene. They found two SNPs rs12206094 and rs4946935 with the strongest association signal to human longevity and confirmed it in several populations. Furthermore, they revealed a possible functional link (binding and enhanced promotor activity) between this two SNPs in FOXO3 and the longevity phenotype in humans. They also stated that these SNPs are associations with increased FOXO3 mRNA expression (156).
Moreover, another human SNP in the FOXO3 gene was recently shown to be associated with an increased risk for malaria but with a milder course in patients with autoimmune diseases like Crohn´s disease and rheumatoid arthritis. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGF-ß1 reduces production of pro-inflammatory cytokines, including TNF-α, and increases production of anti-inflammatory cytokines, including IL-10 (9). In line with that an age-dependent decrease of Foxo3 in old mice and also Foxo3 knockout (KO) mice have been found to induce a loss of anti-inflammatory behavior macrophages (157).
Only two studies provide insight on how variants in the FOXO3 gene might act. Donlon et al. showed by FISH technology, that FOXO3 clusters with neighboring genes after stress activation with H2O2 forming a “transcription complex”. This clustering was stronger in cells with the minor SNP genotype GG (rs2802292) compared with the common genotype TT. This was accompanied by the observation that lymphoid cell lines derived from SNP carriers expressed more FOXO3 mRNA upon induction than cell lines derived from non-carriers (158). Consistently, another study showed that this variant has enhancer function and creates a novel binding site for other transcription factors (159).
1.6 Cardiovascular Disease and FOXO3
Age-associated diseases and ageing of the vasculature system involves increased inflammation accompanied by inflammatory cytokine expression. Loss of microvascular density and plasticity accompanied by dysfunction of stem cells in vascular niches is a major feature of human ageing (101). Deletion of Foxo1, Foxo3 and Foxo4 from myeloid cells in LDL receptor KO mice results in arteriosclerotic plaques that are not only larger, but contain more macrophages having higher proliferative and reduced apoptotic ability (160). These myeloid cells exhibited increased
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Foxo3 plays an important role in cardiac hypertrophy (162, 163), cardiomyocyte survival, differentiation, and remodeling (129). Moreover, Foxo3 plays a critical role in in the resistance to oxidative stress in cardiac fibroblasts (164). Overexpression of FoxO3 in vivo reduces cardiomyocyte size by increasing autophagosome formation, expression of atrogin-1 and the autophagy-related genes encoding microtubule-associated protein 1A/1B-light chain 3, GABA receptor-associated protein-like 1, and autophagy related 12 protein (162). Cardiomyopathy in general comprises a defective heart muscle leading to an impaired systolic pump function. With disease progression, dilatation of the left ventricle occurs in dilated cardiomyopathy (DCM). The resulting phenotype is associated with a poor outcome for the patient even after late onset (165). A subgroup of DCM is termed DCMi, when inflammation is present in the endomyocardium. Based on current concepts, the myocardial inflammation is considered to be elicited by T cells, NK cells, and macrophages amongst other cell types either due to autoimmunity or viral infection (166). Viral infection-triggered myocarditis is associated with high mortality and often needs heart transplantation as therapeutical option (167). Nevertheless, it is not well understood how the immune system recognizes and control viral infection.
1.7 Metformin and salicylate in the immune system
1.7.1 MetforminMetformin (dimethylbiguanide) is a drug used as a first line oral treatment against type 2 diabetes. The biguanide class of antidiabetic medications, which also includes phenformin and buformin, originates from the goat's rue (Galega officinalis) (168). Metformin enters the cell through organic cation transporters (OCT1) and accumulates in the mitochondria where it inhibits complex I. This leads to a reduction in ATP and a linked rise in AMP. Elevated AMP levels lead to activation of AMPK (Figure 4) (169). It has been identified to reduce cardiac events in diabetic patients as well as it improves the overall outcome and prognosis (170) Several studies are now investigating the influence of metformin on aging (targeting/taming aging with metformin, TAME study (171) and Metformin in Longevity Study, MILES study, ClinicalTrials.gov Identifier: NCT02432287). Furthermore, Evans et al reported 2005 in an epidemiological study that the cancer development risk was lower in diabetic patients treated with metformin (172). Antitumor mechanism was believed to be mediated by gluconeogenesis inhibition, reduction
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in insulin concentration and suppressed mTOR (173) via activation of AMPK (174). But not only hypoglycemic properties were described. Metformin also showed anti-inflammatory properties observed in endothelial cells (175), aortic smooth muscle cells (176, 177) and monocyte/macrophage, where less cytokines (IL-1ß and TNF-α) were observed after LPS stimulation (178). Furthermore, murine bone marrow derived macrophages incubated with metformin showed an AMPK dependent reduced inflammatory response (IFN-γ, TNF-α, IL-12) after Legionella Pneumonia infection (179). Besides that, metformin has antioxidative properties by decreasing the NADPH oxidase expression (180) and improving antioxidant activity (glutathione peroxidase, SOD, catalase) in LPS stimulated human monocytes/macrophages (178). Hou et al. discovered that metformin reduces ROS levels by inducing Trx expression through activation of the AMPK-FOXO3 pathway in human aortic endothelial cells (176). Interestingly, Kajiwara et al. showed that metformin induces mitochondrial ROS production and AMPK signaling and enhances thereby the bactericidal activity in macrophages which contribute to the improved survival in L.pneumophilia infection in mice. Metformin induces mitochondrial ROS but not phagosomal NADPH oxidase-derived ROS and gluthathione treatment abolished the positive effect of metformin. (179).
Also Mycobacterium tuberculosis patients had better survival in the metformin treated group (181). Singhal et al. postulated already 2014 that metformin is a potential treatment against intracellular growth of M.tuberculosis partially by increased mitochondrial ROS production (182). Different observations made a characterizing study important.
Gene-based analysis identified seven unique non-synonymous variations in FOXO3, as significantly associated with lower fetal hemoglobin (HbF) in Sickle cell disease. The disease can be ameliorated by increasing HbF levels. FOXO3 is a positive regulator of γ-globin expression, and an excellent therapeutic target for HbF induction. Metformin is a well-studied, well tolerated oral agent, increasing FOXO3 and γ-globin transcription levels in a dose-dependent manner (183).
The maximal approved total daily dose of metformin for treatment of diabetes mellitus is 2.5 g (35 mg/kg body weight). After oral administration of metformin it is rapidly distributed to many tissues with absorbance in the small intestine. The peak plasma concentration occurs in 3 h and
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range from 1-4 mg/l [about 40-70 µM in the portal vein and 10-40 µM in the circulation], with a plasma half-life time of 20 h (184, 185).
Figure 4: Proposed mechanisms of metformin action in diabetes. Metformin enters the cell through OCT1 and accumulates in the mitochondria where it inhibits complex I. This leads to a reduction in ATP and a linked rise in AMP. Elevated AMP levels lead to activation of AMPK, although metformin may also promote AMPK activation in a direct manner. AMPK activation inhibits genes involved in gluconeogenesis, to prevent further energy loss. The further downstream effect of metformin on FOXO3 in immune cells is not elucidated so far and is part of this study. Modified from (169).
1.7.2 Salicylate
Salicylate is an aspirin metabolite that is widely used for the treatment of inflammation-related diseases (186). It is used for reducing pain, fever, inflammation, insulin resistance and protecting against cancer. Possible mechanisms for the pharmacologic actions of salicylate include inhibition of cyclooxygenases and NF-κB (187, 188). A recent report has proposed the idea that all these beneficial effects may not only include inhibition of cyclooxygenases and NF-κB signaling, but also be partly mediated by AMPK activation (188, 189). However, the precise mechanism by which salicylate induces AMPK mediated anti-proliferative and pro-apoptotic effects during inflammation remains unclear (190, 191).
1.8 Aim of this study
FOXO3 is a transcription factor crucial in the regulation of cell metabolism, stress resistance and immunity. Phosphorylation via PI3K/AKT pathway leads to its inactivation and nuclear exclusion whereas AMPK mediated phosphorylation induces activation of FOXO3 (95). Recently, SNPs for FOXO3 have been identified to be associated with longevity (151, 156) and
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favorable outcome in inflammatory disease (9). We could previously show that Foxo3 attenuates NK cell response in a viral myocarditis model (7). The aim of this thesis was to comprehensively elucidate the immunomodulatory role of FOXO3 and its possible therapeutic modulation.
Therefore, we first characterized the impact of Foxo3 on immune cell composition comparing Foxo3-/- mice with wild type (WT) littermates. Further, we analyzed and characterized NK cells phenotypically and functionally in mice. In human NK cells we comparatively analyzed healthy donors carrying SNPs in the FOXO3 gene that are associated with longevity and better outcome in chronic inflammatory diseases. Finally, we investigated if FOXO3 is inducible by metformin and analyzed the involved signaling pathway and influences on ROS/RNS production and cytokine response in human immune cells.
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2 E
XPERIMENTAL PROCEDURES2.1 Material
2.1.1 Ragents and chemicals
If not listed in Table 1 reagents and stimulating agents were purchased by SIGMA-ALDRICH®, Merck Millipore, ROTH® or Serva.
Table 1: Reagents and chemicals
Name Company
5-Aminoimidazole-4-carboxamide riboside (AICAR) Seleckchem
Bicoll Separating Solution Merck Millipore, Biochom
cOmplete™ Roche
Compound C (Dorsomorphin) Abcam®
DEPC-treated water Ambion® life technologieTM
DPBS (1X) Dulbecco’s Phosphate Buffered Saline Gibco® life technologiesTM
DTT Applichem
Ethanol (>96%) Merck Millipore, Biochrom
Fetal Calf Serum (FCS) Merck Millipore, Biochrom
Flebogamma, human IgG Grifols
L-glutamine Merck Millipore, Biochrom
glycine Applichem
human recIL-2 Proleukin® (aldesleukin)
mouse recIL-2 RnD Systems
LPS Enzo®
Methanol (>99,9%) J.T.Baker®
Nonident P 40 (NP-40) Fluka
Oligomycin Seleckchem
Penicillin/ Streptomycin Merck Millipore, Biochrom
paraformaldehyde Science Services
PhosSTOP™ Roche
Resiquimod (R848) Enzo®
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Trypan Blue Gibco® life technologiesTM
Tris Base J.T.Baker®
2.1.2 Antibodies and dyes
Table 2: Primary antibodies -specificity human
Antibody Fluorochrome Clone Company Dilution in 100 µl
FOXO3 pS413 D77C9 Cell signaling 1:100
FOXO3 pan polyclonal Merck Millipore 1:100
CD56 PE HCD56 BioLegend® 1:50
FITC HCD56 BioLegend® 1:50
FITC NCAM16.2 BD Biosciences 1:10
CD3 PB UCHT1 BioLegend® 1:100
AF700 OKT3 BioLegend® 1:40
PerCP Cy5.5 OKT3 BioLegend® 1:100
CD4 AF700 RPA-T4 BioLegend® 1:100
APC-Cy7 SK3 BioLegend® 1:50
CD14 FITC HCD14 BioLegend®® 1:50
CD107a PE H4A3 BD Biosciences 1:5
CD27 BV650 O323 BioLegend® 1:20
IL-10 PE JES3-19F1 BioLegend® 1:20
FoxP3 AF488 259D BioLegend® 1:20
CD25 APC BC96 BioLegend® 1:40
CD3 Per.CP-Cy5.5 OKT3 BioLegend® 1:100
CD14 APC M5E2 BioLegend® 1:50
CD19 PE-Cy7 HIB19 BioLegend® 1:50
CD56 PE HCD56 BioLegend® 1:50
IFN-y AF700 B27 BD Biosciences 1:125
TNF-α PE-Cy7 Mab11 eBioscience 1:200
CD38 FITC HB-7 BioLegend® 1:25
CD24 PerCP-Cy5.5 ML-5 BioLegend® 1:80
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Table 3: Primary antibodies -specificity mouse
Antibody Fluorochrome Clone Company Dilution in 100 µl
CD3 AF700 17A2 Invitrogen 1:100
CD19 APC 6D5 Biolegend® 1:100
Nkp46 APC 29A1.4 Biolegend® 1:100
PE 29A1.4 Biolegend® 1:100
CD11b PE M1/70 eBioscience 1:100
PE-Cy7 M1/70 eBioscience 1:100
CD45 PE-Cy7 30-F11 eBioscience 1:1000
Ly6C PerCP Cy5.5 HK1.4 Invitrogen 1:50
AnnexinV FITC BD Bioscience 1:20
CD69 FITC H1.2F3 eBioscience 1:50
IFN-γ APC XMG1.2 BD Pharmingen 1:100
CD27 APC LG.7F9 eBioscience 1:100
F4/80 AF488 BM8 Biolegend® 1:50
CD11c PE-Cy7 N418 Biolegend® 1:80
CD34 PE RAM34 BD Pharmingen 1:50
Table 4: Secondary antibodies
Antibody Fluorochrome Company Dilution in 100 µl
goat-anti-rabbit AF 700 Invitrogen 1:1000
goat-anti-rabbit HRP Cell signaling 1:1000
goat-anti-rabbit HRP Cell signaling 1:1000
Table 5: Fluorescent dyes
Dye Application Company Dilution in 1 ml
LIVE/DEAD™ Fixable Aqua Live-dead Life Technologies 1:1000 LIVE/DEAD™ Fixable Violet Live-dead Life Technologies 1:1000 2′,7′-Dichlorodihydrofluorescein
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Propidium iodide (PI) Live-dead Invitrogen/Thermo
Fisher 1:500
3,3'-Dioctadecyl-oxacarbocyanine
Perchlorate (DiOC) cytotoxicity Invitrogen/Thermo Fisher 1:250 2.1.3 Cell culture
Table 6: Cell lines
K562 Organism: Homo sapiens
Age: 53 years Gender: female Tissue: bone marrow
Celltype: lymphoblast, chronic myelogenous leukemia Growth properties: suspension
Description: The cells spontaneously differentiate into precursors of the erythroid, granulocytic and monocytic series. The line is EBNA negative.
YAC-1 Organism: Mus musculus, mouse
Strain: A/Sn
Cell Type: Lymphoma/lymphoblast; Moloney murine leukemia virus (MoMuLV) induced
Growth Properties: suspension
Table 7: Media
Medium Additives Company
RPMI 1640 Merck Millipore, Biochrom
IMDM Merck Millipore, Biochrom
Mouse
Medium RPMI supplemented with 1 % glutamine, 1 % Penicillin/Streptomycin and 10 % FCS Human
cultivation medium
IMDM supplemented with 1%
Penicillin/Streptomycin and 10% FCS
Longterm Human cultivation medium supplemented with 100 U/ml human recIL-2
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2.2 Methods
2.2.1 Isolation of mononuclear cells
Fresh PBMC were isolated from heparinized whole blood (vacutainer heparin tubes, BD, Franklin Lakes, USA) by density gradient centrifugation using a Bicoll separating solution. In short, 10 ml whole blood were filled up to a volume of 35 ml with Dulbecco's phosphate-buffered saline (DPBS), mixed and layered onto 15 ml Bicoll separating solution. After 15 min centrifugation at 860 x g, 20 °C without brake, peripheral blood mononuclear cells (PBMCs) were transferred from the interphase to a new tube and washed with an equal volume of DPBS. After 10 min centrifugation at 365 x g, 20°C the supernatant was discarded and the PBMC resuspended in DPBS. Then the cell density was determined using a Neubauer hemocytometer (Laboroptik, Lancing, UK).
2.2.2 Isolation of mouse splenocytes and peripheral blood cells
FVBN WT and FOXO3 KO mice were sacrificed and spleens were taken. Single cell suspensions were made by passing the spleen through a cell strainer and diluting the suspension in cold PBS (4°C). The cells were cultured in RPMI 1640 containing L-glutamine and supplemented with 10% FCS, and 1% each of penicillin and streptomycin at 37°C, 5% CO2, or used immediately. Peripheral blood was obtained by cardial punctuation and collected in heparinized tubes. Blood was diluted with DPBS and separated under sterile conditions by gradient centrifugation using Bicoll with 2000 x g for 15 min without brake. PBMC fraction was collected and used immediately for further analysis.
2.2.3 Detection of AMPK activation using Immunoblot
Signal transduction in human PBMCs cells was performed by 3 h of pre-incubation with metformin and salicylate. The cells were then washed twice in ice cold DPBS and solubilized in lysis buffer for 30 min on ice. After centrifugation at 12000 x g and 4°C for 10 min the supernatant was harvested. Protein concentration was estimated by BCA method (BCA™ Protein Assay Kit Thermo Scientific) according to manufacturer’s instructions (SpectraMax). 20 µg proteins per lane were separated on gradient (7.5%-12.5%) gels with 140 V constant current for about 90 min using Rotiphorese® SDS-PAGE running buffer and transferred to a nitrocellulose membrane by tank blot Bio-Rad® (Bio-Rad Mini Trans-Blot® Cell) using 400 mA
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for 1 h. Precision Plus ProteinTM KaleidoscopeTM (Bio-Rad) was used as standard. First, the membranes were incubated for 1 h in blocking solution. Afterwards membranes were incubated with the primary antibodies mouse-α-GAPDH (1:10000), rabbit-α-AMPK pT172 (1:500), rabbit-α-AMPKalpha (1:500) overnight at 4°C in 5% BSA (in 0.05% TBS-T). After washing 3 times for 10 min in 0.05% TBS-T the membrane was incubated with the secondary antibody goat-α-rabbit or mouse Ig-HRP 1:2000 for 1 h. Following to washing, detection by chemiluminescence was achieved by using an enhanced chemiluminescence system (Pierce™ ECL Western Blotting Substrate) according to the manufacturer’s instructions. Quantification versus GAPDH was performed by ImageJ software (1.48v).
Lysis buffer: 50 mM Tris pH 7.7; 50 mM NaCl; 5 mM MgCl2;freshly added: 1%
NP-40 (v/v); Complete; PhosSTOP; 10 µM MG132
6x SDS sample buffer: 0.35 M Tris pH 6.8; 30% Glycerin (v/v); 10% SDS (w/v); 9.3% DTT
(w/v); 0.006% Bromphenolblau (w/v)
Transfer buffer: 25 mM Tris pH 8.3; 187 mM Glycin; 10% Methanol (v/v)
TBS-T: 50 mM Tris HCl pH 7.9; 154 mM NaCl; 0.1-0.5% (v/v) Tween
Stacking gel: 5% NF-Acrylamide/Bis-solution 30 % (29:1); 0.125 M Tris-HCl pH
6.8; 0.1% SDS (w/v); 0.05% Ammonium persulfate (APS) (v/v); 0.1% TEMED (v/v)
Running gel: NF-Acrylamide/Bis-solution 30 % (29:1) in corresponding
concentrations; 0.376 M Tris-HCl pH 8.8; 0.1% SDS (w/v); 0.05% APS (v/v); 0.8% TEMED (v/v)
2.2.4 Flow cytometry (FC) analysis in human PBMCs FOXO3 activation
For intracellular FOXO3 staining, PBMCs are used after stimulation with Oligomycin [10 µM], Compound C (CC) [10 µM] or metformin [0.1; 1; 10; 20 mM] for 30 min at 37°C. FOXO3 activation is visualized with an unlabeled monoclonal rabbit anti-FOXO3 pS413 antibody and a polyclonal FOXO3 pan antibody. For intracellular staining, the cells are fixed with 2% formalin for 10 min. After a washing step, the pellet is resuspended and permeabilized in 90% methanol
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and are distributed into three FACS tubes. The cells are washed and stained with primary antibodies for FOXO3pan and FOXO3 pS413 for 1 h. After the indicated time the cells are washed, followed by a staining with the secondary AF700-labelled goat-anti-rabbit antibody for 30 min. All staining’s are confirmed using the secondary antibody alone. To avoid cross-reactions with the secondary antibodies, cell surface molecules CD3-PB, CD56-PE, and CD14-FITC are stained afterwards for 15 min at 4°C.
ROS/RNS using DCFH-DA
For intracellular ROS/RNS measurement 1 x 10^6 fresh isolated PBMCs were washed twice with PBS. After stimulating the cells for 3 h with metformin in IBM medium supplemented with 10% FCS and 1% Penicillin/ Streptomycin, cells were washed in centrifuged at 1200 x g for 2 min and resuspended in 100 µl PBS. Cells were extracellularly stained with fluorochrome labeled antibodies for 10 min in the dark. Afterwards, sample volume was adjusted to 1 ml and cells were stimulated with 100 ng/µl of the protein kinase C (PKC) activator Phorbol-12-myristat-13-acetat (PMA) or were treated with 1 mM of H2O2 as positive control for 10 min at 37°C in the incubator. Samples were then centrifuged at 1200 x g for 2 min and supernatants were removed. The cells were washed again with 1 ml of PBS and pelletized at 1200 x g for 2 min. Supernatant was discarded and samples were admitted in 1 ml of PBS. For discrimination between viable and dead cells LIVE/DEAD™ Fixable Violet dead cell stain was added 1:1000. Then, samples were stained with 5 µM of DCFH-DA and incubated for 30 min in the dark at room temperature. After an additional washing step with PBS cells were transferred into FACS tubes and measured on the LSR Fortessa (BD) within 30 min after staining. Analysis was performed using FlowJo V 10.0.8 (Tree Star).
Intracellular cytokine measurement
For intracellular cytokine measurement cells were stimulated with Staphylococcal enterotoxin B (SEB), which binds to MHC class II molecules and specific regions of T cell receptors (TCR), TLR-4 agonist Lipopolysaccharide (LPS), TCR crosslinker Concanavalin A (ConA), TLR-7/8 agonist R848 or with PMA together with the Ca2+ ionophore Ionomycin and incubated for different time periods. Metformin was either coincubated or pre-incubated for different time periods as indicated.
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For stimulation of IFN-γ response of human NK cells complete PBMCs were incubated with R848 [2 µg/ml] for 18 h. Brefeldin A (BFA) [7.5 µg/ml] was added 5 h before analysis. Then IFN-γ staining was performed on total CD56+ (clone NCAM16.2-FITC) and CD56dim and CD56bright NK cells. Cells were washed in FC washing buffer and resuspended in 500 µl PBS containing 1 mM EDTA and incubated for 10 min. After another washing step the cells were stained extracellular (CD3-PB, CD56-PE, LIVE/DEAD™ Fixable Violet) for 30 min at 4°C. Cells were washed with 500 µl BD FACS™ lysing solution (1:10 diluted in PBS) and incubated for 5 min in the dark. After centrifugation, cells were resuspended in 250 µl BD FACS™ Permeabilizing Solution 2 (1:10 diluted in ddH2O) and incubated for 10 min in the dark. 1 ml FC washing buffer was added, centrifuged and intracellular IFN-γ was stained for 30 min at 4°C. After another washing step, cells were measured directly at LSR Fortessa (BD). Analysis was performed using FlowJo V 10.0.8 (Tree Star).
FC washing buffer: 137 mM NaCl; 2.7 mM KCl; 8 mM Na2HPO4 x 2 H2O; 1.5 mM KH2PO4; pH 7.2; 2% Flebo-γ (v/v)
For TNF-α and IFN-γ in human PBMCs 3 ml of a 1x10^6 cells/ml cell suspension were cultured in growth medium containing 100 U/ml IL-2 in a 6-well plate. After 76 h cells were stimulated with ConA [10 µg/ml] for 18 h. BFA [10 µM] was coincubated for 16 h. Cells were harvested and stained for intracellular cytokines as described in the previous section (TNFα-PECy7; IFN-γ AF700).
In order to measure IL-10 expression in regulatory B cells (Bregs), PBMCs were adjusted to 2x10^6 cells/ml in human cultivation medium and 2 ml were added in each well of a 6-well plate. Cells were stimulated with metformin [0.1; 1; 10; 20 mM] + LPS [100 ng/ml] for 48 h at 37 °C and 5% CO2. For the last 6 h PMA [50 ng/ml], Ionomycin [1 µg/ml] and BFA [2.5 µg/ml] were added. Cells were harvested and washed once in FC washing buffer and resuspended and incubated for 10 min in 1 ml DPBS containing 1 mM EDTA. Staining was performed with BD Cytofix/Cytoperm™ according to the protocol. In brief, after another washing step cells were stained extracellular for 30 min in the dark on ice (CD38-FITC; CD24-PerCp-Cy5.5; CD19-PE-Cy7; CD4-APC-Cy7; CD3-AF700; CD27-BV650; CD25-APC; LIVE/DEAD™ Fixable Aqua). Cells were washed in BD Perm/Wash™ Buffer and resuspended in 100 µl Cytofix/Cytoperm™ solution. Following incubation for 20 min at 4°C in the dark, cells were washed twice with washing buffer
