Microglia cells fulfill key homeostatic functions and essentially contribute to host defense within the CNS. Altered activation of microglia, in turn, has been implicated in neuroinflammatory and neurodegenerative diseases. In this study, we identify the nuclear receptor (NR) Nr4a1 as key rheostat controlling the activation threshold and polarization of microglia. In steady-state microglia, ubiquitous neuronal-derived stress signals such as ATP induced expression of this NR, which contributed to the maintenance of a resting and noninflammatory microglia phenotype. Global and microglia-specific deletion of Nr4a1 triggered the spontaneous and overwhelming activation of microglia and resulted in increased cytokine and NO production as well as in an accelerated and exacerbated form of experimental autoimmune encephalomyelitis. Ligand-induced activation of Nr4a1 accordingly ameliorated the course of this disease. Our current data thus identify Nr4a1 as regulator of microglia activation and potentially new target for the treatment of inflammatory CNS diseases such as multiple sclerosis.

Microglia are a highly specialized population of tissue-resident macrophages that fulfill multiple homeostatic functions within the CNS, an organ otherwise void of immune cells (1). These phagocytes closely interact with neurons and their synapses, where they contribute to neurogenesis, brain wiring, and synaptic pruning. Notably, they constantly react to signs of neuronal stress and efficiently remove apoptotic neurons (2, 3). Such microglia-controlled housekeeping functions enable brain development, essentially contribute to the functionality of the adult neuronal network, and facilitate repair mechanisms after brain injury and inflammation-associated tissue damage that occurs during CNS diseases such as experimental autoimmune encephalomyelitis (EAE), where microglia were shown to essentially contribute to the clearance of cellular debris (4).

Upon activation, however, microglia can profoundly change their phenotype, which includes the upregulation of pattern recognition receptors, costimulatory molecules, and proinflammatory cytokines (5). Activated microglia can additionally change their functional polarization and are able to produce large amounts of NO and other reactive oxygen species (6). This series of events eventually results in disruption of the blood–brain barrier, triggers recruitment of nonresident immune cells such as monocytes and T lymphocytes to the CNS, and enables an immune response at this otherwise immune-privileged site (7). Although crucial to prevent spreading of infections within the CNS (8), this process has to be tightly controlled, as an overwhelming or spontaneous activation of microglia and an increased production of NO would result in impaired neuronal function, chronic inflammation, aggravated tissue damage, and neuronal death, all features characteristic of chronic inflammatory CNS diseases such as multiple sclerosis (911). Whereas pathways that contribute to microglial activation have been intensely studied, less is known about factors that stabilize their homeostatic phenotype.

Nuclear receptors (NRs), including the Nr4a subfamily of NRs, coordinate cellular and systemic metabolic processes (12). Recent data suggest that NRs also regulate myeloid cell differentiation as well as their response to inflammatory stimuli (1315). Nr4a family members have been previously implicated as modulators of CNS inflammation, although their exact role in microglia biology remains incompletely understood (16, 17).

Animal experiments were approved by the government of Mittelfranken. Mice were housed in the animal facility of the University of Erlangen–Nuremberg. Nr4a1−/− and Cx3cr1CreER mice were on the C57BL/6 background and obtained from The Jackson Laboratory (Bar Harbor, ME). Nr4a1fl/fl mice were previously described (18). For induction of Cre recombinase, 5- to 7-wk-old Cx3cr1CreER mice were treated with 4 mg of tamoxifen (TAM; Sigma-Aldrich, Munich, Germany) solved in 200 μl of corn oil (Sigma-Aldrich) and injected s.c. at two time points 48 h apart. In all experiments, littermates carrying the respective loxP-flanked alleles but lacking expression of Cre recombinase were used as controls. These mice were used for EAE 4 wk after TAM injection with an age of 8 to 12 wk. For activation of Nr4a1, mice were treated with 12.5 mg/kg body weight cytosporone B (CytB; C2997; Sigma-Aldrich) solved in DMSO and diluted in up to 200 μl of PBS. Mice were injected i.p. every day.

Mice were immunized s.c. with myelin oligodendrocyte glycoprotein peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK) (Charité Berlin, Berlin, Germany) emulsified in CFA (Sigma-Aldrich, which was enriched with Mycobacterium tuberculosis [H37Ra; BD Biosciences, Heidelberg, Germany]) at day 0 to induce EAE. Additionally, 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) was administered i.p. at day 0 and day 2. EAE paralysis of mice was scored as previously described (19). For conditional knockout mice, animals from each group were immunized s.c. 4 wk after TAM injection with an age of 8–12 wk.

Spinal cords of anesthetized and PBS-perfused mice were isolated and fixed in 4% buffered formalin. Tissues were dissected and embedded in paraffin before sectioning. Sections were stained with Luxol Fast Blue using standard procedures. For immunofluorescence, the following Abs were used: anti-Nr4a1 (ab13851; Abcam, Cambridge, U.K.), anti-Iba1 (ab48004; Abcam), donkey anti-rabbit Alexa Fluor 647 (1563697; Life Technologies, Darmstadt, Germany), and donkey anti-goat Alexa Fluor 555 (1458633; Life Technologies).

For quantification of NO2 production by in vitro–generated microglia, cells were incubated with either 1 mM ATP (A6419; Sigma-Aldrich) and/or 100 ng/ml LPS (L-2630; Sigma-Aldrich) or both in combination. Because NO is rapidly converted to NO2 and NO3 in culture medium, NO2 production was quantified using a modified Griess reagent (Sigma-Aldrich) as previously described (20).

Microglia were isolated as previously described (21). Anesthetized mice were perfused intracardially with PBS. Brain and spinal cord were homogenized and isolated cells were separated using a density gradient. After intense washing steps, cells were incubated with Fc Block [TruStain fcX 9(93); BioLegend, London, U.K.] at 4°C for 10 min, followed by surface staining in PBS containing 5% FCS for 30 min at 4°C. Intracellular staining of Nur77 was performed by using a Foxp3/transcription factor staining buffer set (eBioscience, San Diego, CA). Cells were stained according to the manufacturer’s instructions. Microglia were stained with the indicated FACS Abs: Nur77 (12.14, eBioscience), CD39 (Duha59), CD206 (C068C2), Ly6C (HK1.4), Ly6G (1A8), CD86 (2331), MHC class II (L243), CD11b (M1/70), and CD45 (30-F11) (all BioLegend), as well as Live/Dead (Life Technologies). For in vitro experiments, isolated cells were cultured for 3 wk in appropriate DMEM/F12 medium containing 10% FCS and M-CSF to obtain microglia. Following treatment with either 1 mM of ATP (A6419; Sigma-Aldrich) and/or 100 ng/ml LPS (L-2630; Sigma-Aldrich), microglia were further analyzed.

Seven days after immunization with myelin oligodendrocyte glycoprotein peptide, mice were injected i.v. with 150 μl of 4 mg/ml Evans blue (Sigma-Aldrich) solution. Following 4 h of incubation, anesthetized mice were perfused intracardially with 50 ml of ice-cold PBS, and then brains and spinal cords were isolated.

To obtain total cellular tissue RNA, indicated mouse organs were homogenized using Precellys tissue homogenizer (Peqlab, Erlangen, Germany). RNA was extracted using TRIzol reagent (Invitrogen, Darmstadt, Germany). Total RNA was reverse transcribed with human leukemia virus reverse transcriptase using the GeneAmp RNA PCR kit (Applied Biosystems, Darmstadt, Germany) and oligo(dT) primers. Gene expression quantification was performed as previously described (22). The following primer sequences were used: β-actin, 5′-TGT CCA CCT TCC AGC AGA TGT-3′ (sense), 5′-AGC TCA GTA ACA GTC CGC CTA GA-3′ (antisense); arginase-1: 5′-TCA CCT GAG CTT TGA TGT CG-3′ (sense), 5′-CAC CTC CTC TGC TGT CTT CC-3′ (antisense); inducible NO synthase (inos), 5′-CCT TGT TCA GCT ACG CCT TC-3′ (sense), 5′-GCT TGT CAC CAC CAG CAG TA-3′ (antisense); Nr4a1, 5′-CGG ACA GAC AGC CTA AAA GG-3′ (sense), 5′-TAA CGT CCA GGG AAC CAG AG-3′ (antisense).

Protein extracts were isolated from TRIzol phase, and protein concentration was determined with a Bradford assay (5000111; Bio-Rad Laboratories, Hercules, CA). Samples were separated on a 10% SDS–polyacrylamide gel. Proteins were transferred onto a polyvinylidine difluoride membrane and immunoblotted with the following Abs: anti–arginase-1 (sc-18354; Santa Cruz Biotechnology, Dallas, TX); anti-iNOS (a gift of Prof. C. Bogdan and Dr. U. Schleicher), and anti–β-actin (clone AC-74, A2228; Sigma-Aldrich). Quantitative analysis was performed with Photoshop CS5 software, and the results were normalized to the β-actin controls.

Results are expressed as the mean ± SEM. Data were analyzed with a Student t test. A p value <0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). All statistics were performed using GraphPad Prism 6 software.

We previously identified the NR Nr4a1 as key factor orchestrating the noninflammatory clearance of apoptotic cells by resident peritoneal macrophages (14). To determine a potential contribution of Nr4a1 to the homeostatic function of other tissue-resident macrophages, we initially analyzed its expression pattern in different organs, where brain and spinal cord showed by far the highest Nr4a1 mRNA levels during steady-state (Fig. 1A). Immunofluorescence microscopy confirmed the presence of a constitutive Nr4a1 protein expression within the CNS and revealed a particularly high Nr4a1 expression in Iba1+ microglia (Fig. 1B). These data were confirmed by flow cytometry, where we defined microglia as lineage (Lin) (Ly6CLy6GCD206) CD39+ cells of the CNS that could be subdivided into an activated CD45highCD11bhigh (R1) and resting CD45intCD11bhigh (R2) population. We observed that a substantial proportion of both resting and activated microglia expressed Nr4a1 during the steady-state (Fig. 1C). As Nr4a1 is considered to be an inducible NR that is not constitutively expressed (23), these findings were suggestive of the permanent presence of Nr4a1-inducing factors in the microglial environment. Accordingly, we observed a rapid upregulation of Nr4a1 in purified and in vitro–cultivated microglia upon exposure to ubiquitous neuronal-derived stress signals such as ATP (24) (Fig. 1D, Supplemental Fig. 1).

FIGURE 1.

Nr4a1 is expressed in CNS microglia. (A) Nr4a1 mRNA expression in indicated organs and (B) cellular distribution of Nr4a1 protein within the spinal cord of healthy Nr4a1+/+ and Nr4a1−/− mice. Original magnification ×40. (C) Flow cytometry of leukocytes isolated from the CNS of healthy Nr4a1+/+ and Nr4a1−/− mice: left two rows show the gating scheme; right histograms show the expression level of Nr4a1 (Nur77) protein. Numbers in outlined areas indicate percentage cells among live cells (top left plot); positive for side scatter (SSC) and forward scatter (FSC) (top middle plot); CD39+Lin (CD206, Ly6C, Ly6G) cells (top right); and CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells. (D) Nr4a1 mRNA expression in isolated Nr4a1+/+ and Nr4a1−/− microglia after stimulation with 1 mM ATP for the indicated time periods. Data are presented as mean ± SEM. Results are representative of three independent experiments (n = 3). BM, bone marrow; iLN, inguinal lymph node; SC, spinal cord.

FIGURE 1.

Nr4a1 is expressed in CNS microglia. (A) Nr4a1 mRNA expression in indicated organs and (B) cellular distribution of Nr4a1 protein within the spinal cord of healthy Nr4a1+/+ and Nr4a1−/− mice. Original magnification ×40. (C) Flow cytometry of leukocytes isolated from the CNS of healthy Nr4a1+/+ and Nr4a1−/− mice: left two rows show the gating scheme; right histograms show the expression level of Nr4a1 (Nur77) protein. Numbers in outlined areas indicate percentage cells among live cells (top left plot); positive for side scatter (SSC) and forward scatter (FSC) (top middle plot); CD39+Lin (CD206, Ly6C, Ly6G) cells (top right); and CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells. (D) Nr4a1 mRNA expression in isolated Nr4a1+/+ and Nr4a1−/− microglia after stimulation with 1 mM ATP for the indicated time periods. Data are presented as mean ± SEM. Results are representative of three independent experiments (n = 3). BM, bone marrow; iLN, inguinal lymph node; SC, spinal cord.

Close modal

To determine a potential role of Nr4a1 during microglial homeostasis and function, we subsequently performed a phenotypical analysis of microglia in spinal cord and brain of wild-type (WT) and Nr4a1−/− mice during the steady-state. Flow cytometry of the brain of Nr4a1−/− mice revealed an enlarged population of activated CD45highCD11bhigh (R1) and resting CD45intCD11bhigh (R2) microglia that additionally showed an increase in the surface expression of activation markers such as MHC class II and CD86 (Fig. 2A–C). To determine potential alterations in the functional polarization of microglia and to study their potential to generate NO, we additionally measured expression of iNOS and arginase-1, which represent the two enzymes that compete for l-arginine and thereby contribute or interfere with NO production. iNOS protein expression was absent in the CNS of healthy mice, and we detected only small amounts of iNOS mRNA in both WT and Nr4a1−/− mice (Fig. 2D, 2E, Supplemental Fig. 2). Arginase-1 protein and mRNA, in contrast, were constitutively expressed in the healthy CNS of WT mice, but almost absent in Nr4a1−/− mice (Fig. 2D, 2E, Supplemental Fig. 2). Taken together, these data indicated an important role of this NR in the control of the activation threshold as well as the functional polarization of microglia and suggested that Nr4a1 was involved in the regulation of l-arginine metabolism and NO production.

FIGURE 2.

Nr4a1 regulates microglia activation and polarization. (A and B) Flow cytometric analysis of the total frequency of CD39+Lin (CD206, Ly6C, Ly6G), CD45highCD11bhigh (R1), and CD45intCD11bhigh (R2) cells in isolates of healthy Nr4a1+/+ and Nr4a1−/− mice brains. (B and C) Analysis of the activation states of the individual R1 and R2 cell populations via analysis of expression levels of MHC class II (B) and CD86 (C). (D) Western blot analysis of iNOS and arginase-1 protein levels and (E) quantification of inos and arginase-1 mRNA expression levels in extracts of spinal cords isolated from healthy Nr4a1+/+ and Nr4a1−/− mice. Data are presented as mean ± SEM. Results are representative of three independent experiments [n = 3 (D), n = 6 (A–C)]. *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 2.

Nr4a1 regulates microglia activation and polarization. (A and B) Flow cytometric analysis of the total frequency of CD39+Lin (CD206, Ly6C, Ly6G), CD45highCD11bhigh (R1), and CD45intCD11bhigh (R2) cells in isolates of healthy Nr4a1+/+ and Nr4a1−/− mice brains. (B and C) Analysis of the activation states of the individual R1 and R2 cell populations via analysis of expression levels of MHC class II (B) and CD86 (C). (D) Western blot analysis of iNOS and arginase-1 protein levels and (E) quantification of inos and arginase-1 mRNA expression levels in extracts of spinal cords isolated from healthy Nr4a1+/+ and Nr4a1−/− mice. Data are presented as mean ± SEM. Results are representative of three independent experiments [n = 3 (D), n = 6 (A–C)]. *p < 0.05, **p < 0.01, ***p < 0.01.

Close modal

Also, in vitro–cultivated Nr4a1−/− microglia responded with an increased expression of costimulatory molecules after exposure to LPS and endogenous stress signals such as ATP (Fig. 3A). This enhanced activation was paralleled by an increased production of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α (Fig. 3B), which confirmed a key regulatory role of Nr4a1 during microglia activation. Determination of microglia polarization showed that nonstimulated microglia lacked iNOS, but expressed a substantial amount of arginase-1, whereas LPS-induced activation of microglia resulted in the induction of iNOS and the suppression of arginase-1 (Fig. 3C, Supplemental Fig. 3). In line with our in vivo findings, we observed reduced arginase-1 levels in Nr4a1−/− microglia (Fig. 3C, Supplemental Fig. 3). Measurement of microglia-derived NO confirmed a dramatic increase in NO production upon stimulation with LPS. ATP as an Nr4a1-inducing stress signal, in turn, abrogated NO production in WT, but not Nr4a1−/−, microglia (Fig. 3D). These data did not only reveal an inverse regulation of arginase-1 and iNOS expression during microglia activation, but simultaneously suggested an important role of Nr4a1 during the control of l-arginine metabolism and NO production.

FIGURE 3.

Nr4a1 modulates the inflammatory response and NO production of microglia. (AD) In vitro–cultivated microglia that were isolated from CNS of Nr4a1+/+ and Nr4a1−/− mice were stimulated with 1 mM ATP for 2 h before LPS (100 ng/ml) was added for another 14 h. Subsequently, we performed (A) flow cytometric analysis of CD86 expression, (B) ELISA-based measurement of IL-1β, IL-6, and TNF-α as well as (C) Western blot analysis of iNOS and arginase-1 protein levels. (D) Measurement of NO production of Nr4a1+/+ and Nr4a1−/− microglia that were stimulated with 1 mM ATP for 2 h before LPS (100 ng/ml) was added for another 14 h. Data shown are representative of at least three independent experiments (n = 3–5). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 3.

Nr4a1 modulates the inflammatory response and NO production of microglia. (AD) In vitro–cultivated microglia that were isolated from CNS of Nr4a1+/+ and Nr4a1−/− mice were stimulated with 1 mM ATP for 2 h before LPS (100 ng/ml) was added for another 14 h. Subsequently, we performed (A) flow cytometric analysis of CD86 expression, (B) ELISA-based measurement of IL-1β, IL-6, and TNF-α as well as (C) Western blot analysis of iNOS and arginase-1 protein levels. (D) Measurement of NO production of Nr4a1+/+ and Nr4a1−/− microglia that were stimulated with 1 mM ATP for 2 h before LPS (100 ng/ml) was added for another 14 h. Data shown are representative of at least three independent experiments (n = 3–5). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.01.

Close modal

We subsequently addressed a potential regulatory role of Nr4a1 during inflammatory CNS disease. EAE serves as a murine model for human multiple sclerosis and is characterized by a T cell–driven inflammation of the CNS resulting in demyelination and progressive paralysis (25). Microglia contribute both to the initiation and the resolution of this disease, where these cells have been implicated in the control of blood–brain barrier integrity, the regulation of T cell trafficking and T cell activation, as well as postinflammatory repair processes. Furthermore, microglia-derived NO was shown to exert neuronal toxicity during CNS inflammation (6, 26).

Nr4a1 expression was significantly elevated in the CNS of animals suffering from EAE (Fig. 4A). Absence of Nr4a1, in turn, resulted in a dramatic increase in the number of infiltrating myeloid cells as well as of activated microglia during the early prodromal phase of EAE (Fig. 4B). These animals additionally showed an accelerated disruption of the blood–brain barrier (Fig. 4C) and accordingly suffered from a premature onset of clinical symptoms, an exacerbated disease course (Fig. 4D), which was accompanied by a preterm influx of T cells and augmented demyelination (Fig. 4E, Supplemental Fig. 4A). However, Nr4a1−/− mice did not display changes in the composition of their T cell subsets or an increase in the intrinsic proliferative capacity of T cells after Ag encounter (Supplemental Fig. 4B, 4C). Taken together, these data strongly indicated that, during neuroinflammation, Nr4a1 acted as major factor controlling the initial activation of microglia that subsequently impacted the recruitment of T cells into the CNS, but did not intrinsically affect the activation or differentiation of autoreactive T cells. Experiments with mice carrying a microglia-specific deletion of Nr4a1 recapitulated these findings (Fig. 4F). These Cx3Cr1CreER:Nr4a1fl/fl animals again showed a premature onset and increased severity of EAE when compared with their Nr4a1fl/fl littermates, thus confirming a key function of this NR as a microglia rheostat that controlled CNS inflammation.

FIGURE 4.

Expression of Nr4a1 protects from autoimmune-driven CNS inflammation. (A) Nr4a1 mRNA expression levels of extracts isolated from spinal cords and brains of Nr4a1+/+ mice 17 d after induction of EAE (immunized) compared with nonimmunized mice (control). (B) (top) Flow cytometric analysis of the frequency of CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells in the brain of Nr4a1+/+ and Nr4a1−/− mice on day 10 after induction of EAE (gated on live cells); (below) determination of the total frequency of CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells, following exclusion of Ly6G+ neutrophils and CD206+ perivascular macrophages (gating scheme left plots). (C) Evaluation of blood–brain barrier integrity in brains of Nr4a1+/+ and Nr4a1−/− mice on day 7 after induction of EAE, by i.v. administration of Evans blue. (D) Clinical disease course with corresponding time course of body weight and (E) histology of spinal cord sections using Luxol fast blue (LFB) for demyelination from Nr4a1+/+ and Nr4a1−/− mice 17 d after induction of EAE; right, quantification of demyelination. (F) EAE disease course in Cx3cr1CreER:Nr4a1fl/fl mice and their Nr4a1fl/fl littermate controls. Data are presented as mean ± SEM. Data are representative of three (A, B, D, and E), two (F), or one (C) independent experiments [n = 9 mice (D), n = 6 (F)]. *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 4.

Expression of Nr4a1 protects from autoimmune-driven CNS inflammation. (A) Nr4a1 mRNA expression levels of extracts isolated from spinal cords and brains of Nr4a1+/+ mice 17 d after induction of EAE (immunized) compared with nonimmunized mice (control). (B) (top) Flow cytometric analysis of the frequency of CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells in the brain of Nr4a1+/+ and Nr4a1−/− mice on day 10 after induction of EAE (gated on live cells); (below) determination of the total frequency of CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells, following exclusion of Ly6G+ neutrophils and CD206+ perivascular macrophages (gating scheme left plots). (C) Evaluation of blood–brain barrier integrity in brains of Nr4a1+/+ and Nr4a1−/− mice on day 7 after induction of EAE, by i.v. administration of Evans blue. (D) Clinical disease course with corresponding time course of body weight and (E) histology of spinal cord sections using Luxol fast blue (LFB) for demyelination from Nr4a1+/+ and Nr4a1−/− mice 17 d after induction of EAE; right, quantification of demyelination. (F) EAE disease course in Cx3cr1CreER:Nr4a1fl/fl mice and their Nr4a1fl/fl littermate controls. Data are presented as mean ± SEM. Data are representative of three (A, B, D, and E), two (F), or one (C) independent experiments [n = 9 mice (D), n = 6 (F)]. *p < 0.05, **p < 0.01, ***p < 0.01.

Close modal

Although Nr4a1 is primarily regulated at the level of expression, and no endogenous ligand for this NR has been identified yet, the natural occurring octaketide CytB was shown to specifically induce transcriptional activity of Nr4a1 (27). We consequently evaluated the potential of Nr4a1 as therapeutic target for inflammatory CNS diseases and performed a CytB treatment during EAE, which indeed resulted in a significantly attenuated disease course in WT, but not in Nr4a1−/−, mice (Fig. 5A, 5B). This beneficial effect of CytB was accompanied by a decreased infiltration of myeloid cells, a reduced number of activated microglia, a diminished microglial activation state, as well as by a protection from demyelination (Fig. 5C, 5D).

FIGURE 5.

Therapeutic targeting of Nr4a1 ameliorates disease course of EAE. (A) EAE disease course of Nr4a1+/+ and (B) Nr4a1−/− mice that received a daily i.p. dose of CytB (12.5 mg/kg body weight) and a vehicle, respectively. (C) Flow cytometry analysis of leukocytes isolated from the CNS of Nr4a1+/+ mice 16 d after induction of EAE that either were treated with CytB or DMSO. Top row: frequency of CD45highCD11bneg invading leukocytes, CD45highCD11bhigh (R1), and CD45intCD11bhigh (R2) cells. Two bottom rows: frequency of total CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells, excluding Ly6G+ neutrophils and Ly6C+ monocyte-derived cells (gating scheme left middle plot) and corresponding quantification (right). Right: histograms and quantification of MHC class II expression (mean fluorescence intensity [MFI]) on R1 and R2. (D) Histology of spinal cord sections using Luxol fast blue (LFB) for assessment of demyelination from CytB- or DMSO-treated Nr4a1+/+ mice 16 d after induction of EAE. Right: quantification of demyelination. Scale bars, 200 μm (left), 100 μm (right). Data are presented as mean ± SEM. Data are representative of three independent experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 5.

Therapeutic targeting of Nr4a1 ameliorates disease course of EAE. (A) EAE disease course of Nr4a1+/+ and (B) Nr4a1−/− mice that received a daily i.p. dose of CytB (12.5 mg/kg body weight) and a vehicle, respectively. (C) Flow cytometry analysis of leukocytes isolated from the CNS of Nr4a1+/+ mice 16 d after induction of EAE that either were treated with CytB or DMSO. Top row: frequency of CD45highCD11bneg invading leukocytes, CD45highCD11bhigh (R1), and CD45intCD11bhigh (R2) cells. Two bottom rows: frequency of total CD45highCD11bhigh (R1) and CD45intCD11bhigh (R2) cells, excluding Ly6G+ neutrophils and Ly6C+ monocyte-derived cells (gating scheme left middle plot) and corresponding quantification (right). Right: histograms and quantification of MHC class II expression (mean fluorescence intensity [MFI]) on R1 and R2. (D) Histology of spinal cord sections using Luxol fast blue (LFB) for assessment of demyelination from CytB- or DMSO-treated Nr4a1+/+ mice 16 d after induction of EAE. Right: quantification of demyelination. Scale bars, 200 μm (left), 100 μm (right). Data are presented as mean ± SEM. Data are representative of three independent experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.01.

Close modal

NRs such as peroxisomal proliferator–activated receptors were initially identified as key factors controlling energy homeostasis and are targets of drugs that have been already approved for the treatment of diseases such as type 2 diabetes or hyperlipidemia (28). However, increasing evidence suggests an additional key role of various NRs during the regulation of the innate and adaptive immune response (29). Also, Nr4a1 has been implicated as a pleiotropic regulator of homeostatic processes throughout the body, which include glucose and fat metabolism (30) as well as the differentiation of monocyte subsets (13). Furthermore, Nr4a1 was shown to control the activation and polarization of macrophages, thereby exerting beneficial effects during different disease settings such as atherosclerosis and systemic lupus (14, 31). The current identification of Nr4a1 as an intrinsic modulator of microglia activation opens potential new avenues for the treatment of various neuroinflammatory diseases. These data on a protective role of Nr4a1 expression during EAE are in line with a recent report showing that this NR suppresses the production of norepinephrine in macrophages and thereby limits CNS inflammation after passive transfer of encephalogenic T cells (32). Our present findings suggest that Nr4a1 contributes to the maintenance of a microglia activation threshold that prevents spontaneous and overwhelming activation as well as chronic CNS inflammation. Moreover, Nr4a1 impacts the functional polarization of microglia and regulates arginase-1 expression. Arginase-1 competes with iNOS for l-arginine and thus interferes with NO production (33). Although it will be interesting to delineate the molecular pathways and transcriptional events that mediate regulation of arginase-1 by Nr4a1, our data suggest that this pathway negatively controls NO production by microglia and might thus prevent microglia-induced neurotoxicity. During CNS inflammation, resident and monocyte-derived microglia are not only the first immune cells on site, but they are also central players during the consecutive resolution of the inflammatory response. Although these facts render microglia highly attractive targets for therapeutic intervention during inflammatory degenerative CNS diseases, current treatment strategies hardly affect this cellular compartment (34). Our present study, in turn, shows the feasibility of therapeutically modulating the microglia response during CNS inflammation. Future treatment strategies for humans suffering from related diseases might thus target cells of the adaptive immune system and microglia in parallel to increase the therapeutic response.

We thank C. Stoll, A. Klej, B. Happich and H. Symowski for excellent technical assistance.

This work was supported by Interdisciplinary Center for Clinical Research Erlangen Grant IZKF A55 (to G.K.), Deutsche Forschungsgemeinschaft doctoral training program Grants GRK1660, SPP1468-IMMUNOBONE, and CRC1181 (to G.K.), Else-Kröner Fresenius Stiftung Grant 2013_A274 (to G.K.), and by European Union Grant European Research Council Starting Grants 640087–SOS (to G.K.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

CytB

cytosporone B

EAE

experimental autoimmune encephalomyelitis

iNOS

inducible NO synthase

Lin

lineage

NR

nuclear receptor

TAM

tamoxifen

WT

wild-type.

1
Waisman
A.
,
Ginhoux
F.
,
Greter
M.
,
Bruttger
J.
.
2015
.
Homeostasis of microglia in the adult brain: review of novel microglia depletion systems.
Trends Immunol.
36
:
625
636
.
2
Parkhurst
C. N.
,
Gan
W. B.
.
2010
.
Microglia dynamics and function in the CNS.
Curr. Opin. Neurobiol.
20
:
595
600
.
3
Parkhurst
C. N.
,
Yang
G.
,
Ninan
I.
,
Savas
J. N.
,
Yates
J. R.
 III
,
Lafaille
J. J.
,
Hempstead
B. L.
,
Littman
D. R.
,
Gan
W. B.
.
2013
.
Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.
Cell
155
:
1596
1609
.
4
Yamasaki
R.
,
Lu
H.
,
Butovsky
O.
,
Ohno
N.
,
Rietsch
A. M.
,
Cialic
R.
,
Wu
P. M.
,
Doykan
C. E.
,
Lin
J.
,
Cotleur
A. C.
, et al
.
2014
.
Differential roles of microglia and monocytes in the inflamed central nervous system.
J. Exp. Med.
211
:
1533
1549
.
5
Wu
W.
,
Shao
J.
,
Lu
H.
,
Xu
J.
,
Zhu
A.
,
Fang
W.
,
Hui
G.
.
2014
.
Guard of delinquency? A role of microglia in inflammatory neurodegenerative diseases of the CNS.
Cell Biochem. Biophys.
70
:
1
8
.
6
Brown
G. C.
,
Neher
J. J.
.
2010
.
Inflammatory neurodegeneration and mechanisms of microglial killing of neurons.
Mol. Neurobiol.
41
:
242
247
.
7
Prinz
M.
,
Mildner
A.
.
2011
.
Microglia in the CNS: immigrants from another world.
Glia
59
:
177
187
.
8
Ousman
S. S.
,
Kubes
P.
.
2012
.
Immune surveillance in the central nervous system.
Nat. Neurosci.
15
:
1096
1101
.
9
Dendrou
C. A.
,
Fugger
L.
,
Friese
M. A.
.
2015
.
Immunopathology of multiple sclerosis.
Nat. Rev. Immunol.
15
:
545
558
.
10
Lassmann
H.
,
van Horssen
J.
.
2011
.
The molecular basis of neurodegeneration in multiple sclerosis.
FEBS Lett.
585
:
3715
3723
.
11
van Horssen
J.
,
Witte
M. E.
,
Schreibelt
G.
,
de Vries
H. E.
.
2011
.
Radical changes in multiple sclerosis pathogenesis.
Biochim. Biophys. Acta
1812
:
141
150
.
12
Kurakula
K.
,
Koenis
D. S.
,
van Tiel
C. M.
,
de Vries
C. J.
.
2014
.
NR4A nuclear receptors are orphans but not lonesome.
Biochim. Biophys. Acta
1843
:
2543
2555
.
13
Hanna
R. N.
,
Carlin
L. M.
,
Hubbeling
H. G.
,
Nackiewicz
D.
,
Green
A. M.
,
Punt
J. A.
,
Geissmann
F.
,
Hedrick
C. C.
.
2011
.
The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C monocytes.
Nat. Immunol.
12
:
778
785
.
14
Ipseiz
N.
,
Uderhardt
S.
,
Scholtysek
C.
,
Steffen
M.
,
Schabbauer
G.
,
Bozec
A.
,
Schett
G.
,
Krönke
G.
.
2014
.
The nuclear receptor Nr4a1 mediates anti-inflammatory effects of apoptotic cells.
J. Immunol.
192
:
4852
4858
.
15
Glass
C. K.
,
Saijo
K.
.
2010
.
Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells.
Nat. Rev. Immunol.
10
:
365
376
.
16
Shaked
I.
,
Hanna
R. N.
,
Shaked
H.
,
Chodaczek
G.
,
Nowyhed
H. N.
,
Tweet
G.
,
Tacke
R.
,
Basat
A. B.
,
Mikulski
Z.
,
Togher
S.
, et al
.
2015
.
Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.
Nat. Immunol.
16
:
1228
1234
.
17
Saijo
K.
,
Winner
B.
,
Carson
C. T.
,
Collier
J. G.
,
Boyer
L.
,
Rosenfeld
M. G.
,
Gage
F. H.
,
Glass
C. K.
.
2009
.
A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death.
Cell
137
:
47
59
.
18
Sekiya
T.
,
Kashiwagi
I.
,
Yoshida
R.
,
Fukaya
T.
,
Morita
R.
,
Kimura
A.
,
Ichinose
H.
,
Metzger
D.
,
Chambon
P.
,
Yoshimura
A.
.
2013
.
Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis.
Nat. Immunol.
14
:
230
237
.
19
Rothe
T.
,
Gruber
F.
,
Uderhardt
S.
,
Ipseiz
N.
,
Rössner
S.
,
Oskolkova
O.
,
Blüml
S.
,
Leitinger
N.
,
Bicker
W.
,
Bochkov
V. N.
, et al
.
2015
.
12/15-Lipoxygenase-mediated enzymatic lipid oxidation regulates DC maturation and function.
J. Clin. Invest.
125
:
1944
1954
.
20
Miranda
K. M.
,
Espey
M. G.
,
Wink
D. A.
.
2001
.
A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite.
Nitric Oxide
5
:
62
71
.
21
Lee
J. K.
,
Tansey
M. G.
.
2013
.
Microglia isolation from adult mouse brain.
Methods Mol. Biol.
1041
:
17
23
.
22
Krönke
G.
,
Bochkov
V. N.
,
Huber
J.
,
Gruber
F.
,
Blüml
S.
,
Fürnkranz
A.
,
Kadl
A.
,
Binder
B. R.
,
Leitinger
N.
.
2003
.
Oxidized phospholipids induce expression of human heme oxygenase-1 involving activation of cAMP-responsive element-binding protein.
J. Biol. Chem.
278
:
51006
51014
.
23
McMorrow
J. P.
,
Murphy
E. P.
.
2011
.
Inflammation: a role for NR4A orphan nuclear receptors?
Biochem. Soc. Trans.
39
:
688
693
.
24
Koizumi
S.
,
Ohsawa
K.
,
Inoue
K.
,
Kohsaka
S.
.
2013
.
Purinergic receptors in microglia: functional modal shifts of microglia mediated by P2 and P1 receptors.
Glia
61
:
47
54
.
25
Croxford
A. L.
,
Kurschus
F. C.
,
Waisman
A.
.
2011
.
Mouse models for multiple sclerosis: historical facts and future implications.
Biochim. Biophys. Acta
1812
:
177
183
.
26
Goldmann
T.
,
Prinz
M.
.
2013
.
Role of microglia in CNS autoimmunity.
Clin. Dev. Immunol.
2013
:
208093
.
27
Zhan
Y.
,
Du
X.
,
Chen
H.
,
Liu
J.
,
Zhao
B.
,
Huang
D.
,
Li
G.
,
Xu
Q.
,
Zhang
M.
,
Weimer
B. C.
, et al
.
2008
.
Cytosporone B is an agonist for nuclear orphan receptor Nur77.
Nat. Chem. Biol.
4
:
548
556
.
28
Evans
R. M.
,
Barish
G. D.
,
Wang
Y. X.
.
2004
.
PPARs and the complex journey to obesity.
Nat. Med.
10
:
355
361
.
29
Huang
W.
,
Glass
C. K.
.
2010
.
Nuclear receptors and inflammation control: molecular mechanisms and pathophysiological relevance.
Arterioscler. Thromb. Vasc. Biol.
30
:
1542
1549
.
30
Pearen
M. A.
,
Muscat
G. E.
.
2010
.
Minireview: nuclear hormone receptor 4A signaling: implications for metabolic disease.
Mol. Endocrinol.
24
:
1891
1903
.
31
Hanna
R. N.
,
Shaked
I.
,
Hubbeling
H. G.
,
Punt
J. A.
,
Wu
R.
,
Herrley
E.
,
Zaugg
C.
,
Pei
H.
,
Geissmann
F.
,
Ley
K.
,
Hedrick
C. C.
.
2012
.
NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis.
Circ. Res.
110
:
416
427
.
32
Shaked
I.
,
Hanna
R. N.
,
Shaked
H.
,
Chodaczek
G.
,
Nowyhed
H. N.
,
Tweet
G.
,
Tacke
R.
,
Basat
A. B.
,
Mikulski
Z.
,
Togher
S.
, et al
.
2015
.
Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.
Nat. Immunol.
16
:
1228
1234
.
33
Mori
M.
,
Gotoh
T.
.
2000
.
Regulation of nitric oxide production by arginine metabolic enzymes.
Biochem. Biophys. Res. Commun.
275
:
715
719
.
34
Rangarajan
P.
,
Eng-Ang
L.
,
Dheen
S. T.
.
2013
.
Potential drugs targeting microglia: current knowledge and future prospects.
CNS Neurol. Disord. Drug Targets
12
:
799
806
.

The authors have no financial conflicts of interest.

Supplementary data