Dendritic cells (DCs) function is intimately linked to microenvironment and metabolism. Type I interferons (IFNs) condition dendritic cells to respond to weak self-signals, leading to autoimmunity. However, the metabolic adaption in the process is unclear. Here, we identified spermidine as a critical metabolite impacting the metabolic fitness of DC. First, dynamic metabolome screening indicated that spermidine decreased during IFN priming and following TLR7 ligand stimulation, accompanied by metabolic change from oxidative phosphorylation to glycolysis. Second, spermidine supplement restrained the glycolysis and prevented the overactivation of IFN-a primed DC both in vivo and in vitro. Third, mechanism study uncovered that the activity of FOXO3 adapted to the metabolic change, mediating the anti-inflammatory effect of spermidine. More importantly, addition of spermidine in vivo greatly alleviated the development of psoriasis-like symptom in mice. Thus, our studies revealed metabolic changes boosting DC responses and identified spermidine as a potential therapeutic agent for autoimmune diseases.

Dendritic cells (DCs) are a distinct lineage of mononuclear phagocyte, specialized in antigen presentation to T cells and the control of immunity. Multiple subtypes of DC that have distinct functions and molecular characteristics have been identified (Durai and Murphy, 2016; Malissen et al., 2014; Merad et al., 2013; Shortman and Naik, 2007; Worbs et al., 2017). There are three major subsets of DCs: plasmacytoid DCs (pDCs), conventional DCs (cDCs), and monocyte-derived DCs (moDCs). PDCs are a distinct lineage of DCs that are more specialized for cytokine production, particularly type I interferon (IFN) production, rather than antigen presentation. CDCs within non-lymphoid tissue are composed of two major subpopulations, which are distinguished by the expression of CD103 or CD11b, migrating to lymphoid node and maintaining homeostasis in the steady state (Merad et al., 2013; Shortman and Naik, 2007). In the inflammatory state, monocytes can infiltrate lesion sites and develop into inflammatory myeloid DCs (mDCs) producing tumor necrosis factor (TNF)-a, interleukin (IL)-12, and IL-23 with the activation of both T helper (Th)1 and Th17 cells (Malissen et al., 2014; Worbs et al., 2017). It is known from a wide range of studies that type I IFNs alone or in combination with other cytokines and growth factors such as granulocyte/monocyte colony-stimulating factor (GM-CSF) promote the differentiation and activation of DCs derived from monocytes (Blanco et al., 2001; Farkas and Kemeny, 2012; Postat et al., 2018; Rodriguez-Pla et al., 2014; Santini et al., 2011).IFN-a effectively abrogates Toll-like receptor (TLR)-induced cross-tolerance by priming chromatin to enable moDC to produce robust transcriptional responses to weak signals, leading to the immune response out of control.

Furthermore, IFN-a primed moDCs (IFN-DCs) upregulate TLR7 expression and response to self-RNA, which is absent in GM-CSF-induced moDCs (Mohty et al., 2003). Therefore, IFNDCs are considered to be critical for the pathology of autoimmune disorders such as systemic lupus erythematosus (SLE) and psoriasis (Ganguly, 2018).It is becoming increasingly clear that different stages of DC activation coincide with different types of cellular metabolism to satisfy the bioenergetic and biosynthetic needs of these cells (Everts and Pearce, 2014; O’Neill and Pearce, 2016; Pearce and Everts, 2015; Postat et al., 2018; Wu et al., 2016). Under noninflammatory conditions, catabolic metabolism in DCs centered on mitochondrial oxidative phosphorylation (OXPHOS) is associated with cellular longevity and quiescence. Upon TLR agonists stimulation, DCs undergo a robust metabolic switch characterized by an increase in glycolysis and a progressive loss of OXPHOS signaling (Everts and Pearce, 2014; O’Neill and Pearce, 2016; Pearce and Everts, 2015). Pathways regulating these metabolic states have been uncovered. The conserved kinase mammalian/mechanistic target of rapamycin (mTOR) and its upstream activators phosphoinositide 3-kinase (PI3K)-Akt have been identified as central regulators promoting glycolysis and anabolic metabolism, whereas AMP Kinase (AMPK) is found antagonizing biosynthetic pathways and promoting mitochondrial biogenesis to increase mitochondrial OXPHOS (Everts and Pearce, 2014; O’Neill and Pearce, 2016; Pearce and Everts, 2015). Balance between mTOR and AMPK determines the TLR-induced glucose consumption, mitochondrial OXPHOS, and thereby the immunogenic properties of DC (Everts and Pearce, 2014; O’Neill and Pearce, 2016; Pearce and Everts, 2015).

Metabolic reprograming of DCs is particularly crucial in autoimmune responses because nutrients often change dramatically in autoimmune patients (Everts and Pearce, 2014). In addition to the proven epigenetic regulation (Park et al., 2017), cytokines, such as type I IFNs, in the self-reactive environment may also affect the metabolic state of DC, thus making it more easily activated. Emerging studies have revealed molecules and pathways involved in the aberrant activation of DC upon IFN priming. However, few studies clarify the metabolic changes in the process. Here, we screened the change of amino acids and their derivates in DC during IFN-priming and TLR7-activating stages. In particular, the levels of spermidine decreased continuously in both processes. Meanwhile, IFN-a priming and TLR7 ligand stimulation skewed the metabolism of DC from mitochondrial OXPHOS toward glycolysis. Meanwhile, the AMPK pathway was inhibited and the mTORC1 pathway was activated. Addition of spermidine reversed the process and prevented DC activation. Mechanism study further identified that the phosphorylation sites of FOXO3 changed with the metabolic status and mediated the anti-inflammatory effect of spermidine at the transcriptional factor level. Genetic deletion of foxo3a weakened the function of spermidine. More importantly, supplement of spermidine alleviated the psoriasis-like symptoms and systematic inflammation in an imiquimod (IMQ)-induced psoriasis-like mouse model. Therefore, our results highlight spermidine as a key metabolite in DCs, necessary to maintain normal immune responses and treat autoimmune diseases.

 L-arginine metabolic pathway consists of the L-arginine-nitric oxide (NO) pathway and polyamine catabolism pathway (Babicova et al., 2011; Locke et al., 2016; Mondanelli et al., 2017). As the major components of polyamine catabolism (Locke et al., 2016), both spermidine and spermine decreased during IFN priming and following TLR7 ligand stimulation (Figures 1B and 1C). On the contrary, L-citrulline, major metabolic intermediates of L-arginine-NO pathway (Locke et al., 2016), increased from 4 to 16 h of TLR7 ligand stimulation. L-arginine showed no obvious change in the IFN-priming stage but increased significantly in the IFN-DC maturation stage (Figures 1B and 1C). It indicated that L-arginine was mainly directed to the L-arginine-NO pathway at the expense of polyamine synthesis. The changes in metabolic pathways may be adapted to the changes in DC function. Meanwhile, we detected the expression of enzymes (Geiger et al., 2016; Locke et al., 2016). IFN-a priming led to reduction of enzymes controlling polyamine synthesis, including arginase 1 (Arg1), ornithine decarboxylase 1 (Odc1), spermidine synthase (Srm), and spermine synthase (Sms). In contrast, Nos2, which catalyzes the production of NO from L-arginine, was induced by IFN-a, significantly. During TLR7 ligand stimulation, Srm and Odc1 decreased, whereas Nos2 and Sms increased (Figure 1D), leading to the further reduction of spermidine and increase of L-citrulline. The changes in the mRNA levels of these metabolic enzymes were consistent with changes in metabolites. Therefore, L-arginine and its derivatives might be more critical for the pro-inflammatory function of IFN-DC.

Under conditions of high glucose, AKT phosphorylates FOXO3 at three conserved residues, Thr32, Ser253, and Ser315. This leads to FOXO3 binding to 14-3-3, exposure of the nuclear localization signal, transporting to the cytosol and being degraded, causing reduced expression of FOXO3 target genes (Zhang et al., 2018). When ATP is low, FOXO3 is phosphorylated by AMPK at Ser413 and exhibits increased activity (Lee et al., 2013; Zhang et al., 2018). Foxo3a-deficient DCs express higher levels of proinflammatory cytokines (Dejean et al., 2009; Lee et al., 2013; Litvak et al., 2012; Thompson et al., 2015; Zhang et al., 2018). For these reasons, we hypothesized that FOXO3 might be the critical transcriptional factor in the process.To test our hypothesis, moDCs were stimulated with IFN-a and IFN-DCs were stimulated with R837, together with spermidine or not. The phosphorylation levels of AKT and FOXO3 were detected. On the one hand, AKT was activated by IFN priming and TLR ligand stimulation, whereas the activation was hampered by spermidine (Figure 4B). Then, activated AKT phosphorylates FOXO3 at Ser253, whereas it was suppressed by spermidine (Figure 4B). On the other hand, the activated form of FOXO3 phosphorylated at Ser413 decreased during IFN priming, accompanied by suppressed AMPK signaling (Figures 3C and 4B). By contrast, spermidine restored the activity of AMPK pathway and induced increased phosphorylation levels of FOXO3 at Ser413 in the presence of R837 (Figures 3C and 4B). Therefore, spermidine induced the activated form of FOXO3 (p-FOXO3 at Ser413) and reduced the inactive form of FOXO3 (pFOXO3 at Ser253) during IFN-DC activation (Figure S4A and S4B). 

The end result was that spermidine caused FOXO3 to localize in the nucleus where it was active, as we demonstrated by separating the cytoplasmic and nuclear fractions of DCs, and finding that spermidine increased the amount of FOXO3 in the nucleus (Figure 4C). Finally, the activation of NF-kB was suppressed by spermidine (Figure 4B).To verify that FOXO3 mediates the anti-inflammatory effect of spermidine, moDCs derived from WT or Foxo3adeficient mice were primed with IFN-a for 24 h, and then cultured with R837, together with or without spermidine for 24 h, respectively. The knockout efficiency has been confirmed (Figure S4C). Compared with control, Foxo3adeficient IFN-DCs showed higher expression levels of TNF-a, IL-6, and IL-12/23 p40. Addition of spermidine led to a reduction of 63.8%, 72.6%, and 70.8% in the expression of TNF-a, IL-6, and IL12/23 p40 in wild-type IFN-DCs, whereas only 18.8%, 26.9%, and 16.1% reduction were detected in Foxo3a
/
IFN-DCs, respectively (Figure 4D). Thus, the anti-inflammatory effect of spermidine was greatly weakened in Foxo3a
/
IFN-DCs. 

The role of type I IFNs and TLR7 has been studied more extensively in IMQ-induced psoriatic mice model than in other autoimmune disease models. TLR7 is essential for the full progression of psoriasis during the 6 days induction, whereas the type I IFNs are largely independent of plaque formation (Wohn et al., 2013). However, auto- and paracrine signals via type I IFN are essential to mediate the innate systemic proinflammatory cytokine response after topical IMQ application, as induction of the early systemic proinflammatory cytokines IFN-a, IL-22, and IL-6 was found significantly lower in IFNAR
/
mice (Wohn et al., 2013). The synergistic effect of IMQ and IFN-a was proven to cause the activation of moDCs, leading to the production of TNF-a, IL-6, and IL-23 required for Th17 differentiation (Ueyama et al., 2014). Therefore, the regulation of spermidine on the function of IFN-DC in vivo can be evaluated by detecting skin plaque formation and systematic inflammation including splenomegaly and cytokine production in serum. Repetitive application of IMQ onto wild-type mouse skin led to psoriasiform inflammation with significant thickening, redness, and scaling caused by keratinocyte hyperproliferation and leukocyte infiltration into the skin (Figures 5A–5C). High levels of IFN-a were detected as early as 8 h post first IMQ application when the mRNA levels of TLR7 remained low 2 days after induction (Figure S5A and S5B). The result that expression of TLR7 was significantly induced in dermal moDCs 4 days after induction suggests that the skin lesion sites might experience a local IFN-a priming process. By contrast, mice injected intraperitoneally with spermidine showed less skin inflammation, exhibiting less thickening, redness, and scaling (Figures 5B and 5C).