The Journal of Immunology
The Nuclear Receptor Nr4a1 Mediates Anti-Inflammatory Effects of Apoptotic Cells Natacha Ipseiz,*,† Stefan Uderhardt,*,† Carina Scholtysek,*,† Martin Steffen,*,† Gernot Schabbauer,‡ Aline Bozec,*,† Georg Schett,* and Gerhard Kro¨nke*,† Uptake of apoptotic cells (ACs) by macrophages ensures the nonimmunogenic clearance of dying cells, as well as the maintenance of self-tolerance to AC-derived autoantigens. Upon ingestion, ACs exert an inhibitory influence on the inflammatory signaling within the phagocyte. However, the molecular signals that mediate these immune-modulatory properties of ACs are incompletely understood. In this article, we show that the phagocytosis of apoptotic thymocytes was enhanced in tissue-resident macrophages where this process resulted in the inhibition of NF-kB signaling and repression of inflammatory cytokines, such as IL-12. In parallel, ACs induced a robust expression of a panel of immediate early genes, which included the Nr4a subfamily of nuclear receptors. Notably, deletion of Nr4a1 interfered with the anti-inflammatory effects of ACs in macrophages and restored both NF-kB signaling and IL12 expression. Accordingly, Nr4a1 mediated the anti-inflammatory properties of ACs in vivo and was required for maintenance of self-tolerance in the murine model of pristane-induced lupus. Thus, our data point toward a key role for Nr4a1 as regulator of the immune response to ACs and of the maintenance of tolerance to “dying self.” The Journal of Immunology, 2014, 192: 4852–4858.
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acrophages (MFs) critically contribute to the immunologically silent disposal of apoptotic cells (ACs). A failure in this process eventually results in an aberrant immune response to AC-derived self-Ags, such as dsDNA or histones, as well as in severe autoimmune diseases like systemic lupus erythematosus (1, 2). Notably, ACs are not only silently removed, but their binding and uptake also actively repress the expression of proinflammatory cytokines within the MFs. However, the molecular mechanisms underlying such anti-inflammatory properties of ACs are still incompletely understood (3). Nuclear receptors (NRs) make up a superfamily of ligandactivated transcription factors that are able to recruit both transcriptional coactivators and corepressors and, thereby, positively and negatively affect transcription in a promoter-specific manner. Although NRs initially were identified as key regulators of fat and glucose metabolism (4), recent data highlighted an additional role for NRs during the regulation of the innate and adaptive immune response (5). The Nr4a subfamily of NRs consists of three members (Nr4a1– 3), which are rapidly induced in response to stimuli, such as LPS and oxidized lipoproteins. Nr4a family members were shown to
*Department of Internal Medicine 3 and Institute for Clinical Immunology, Erlangen, Erlangen 91054, Germany; †Nikolaus Fiebiger Center of Molecular Medicine, University Hospital Erlangen, Erlangen 91054, Germany; and ‡Institute for Physiology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna 1090, Austria Received for publication December 18, 2013. Accepted for publication March 17, 2014. This work was supported by the Deutsche Forschungsgemeinschaft (KR3523 and SPP1468-IMMUNOBONE), the MASTERSWITCH and IMI projects of the European Union, the Interdisciplinary Centre for Clinical Research and ELAN-Fonds of the University of Erlangen, and the Bayrische Forschungsstiftung. Address correspondence and reprint requests to Prof. Gerhard Kro¨nke, University of Erlangen, Glueckstrasse 4a, Erlangen 91054, Germany. E-mail address:
[email protected] The online version of this article contains supplemental material. Abbreviations used in this article: AC, apoptotic cell; CHX, cycloheximide; MF, macrophage; NR, nuclear receptor; PS, phosphatidylserine; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303377
exert transcriptional activity in a ligand-independent manner (6), and they have been implicated in the regulation of glucose metabolism, apoptosis, and inflammation, as well as the differentiation of monocytes and T cells (7–14). Our present data show that the binding and uptake of ACs by tissue-resident MFs resulted in the rapid induction of Nr4a1, which, in turn, interfered with NF-kB signaling and the expression of proinflammatory genes, such as IL-12. Furthermore, we provide evidence that this pathway contributes both to the noninflammatory disposal of ACs in vivo and to the maintenance of immunological tolerance to self-Ags.
Materials and Methods Mice Animal experiments were approved by the government of Mittelfranken. Mice were housed in the animal facility of the University of ErlangenNuremberg. All mice used were on the C57BL/6 background. NR4a12/2 mice were on the C57BL/6 background and were obtained from The Jackson Laboratory.
Peritoneal MFs Resident MFs were isolated from the peritoneum of naive 8–10-wk-old mice. Inflammatory MFs were isolated from peritonitis exudates, after i.p. injection of 3% Brewer’s thioglycollate (Sigma-Aldrich) into wild-type (WT) and NR4a12/2 mice at the age of 8–10 wk. Both elicited and resident peritoneal MFs were obtained by peritoneal lavage with ice-cold 4% FCS/PBS and cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. To purify the elicited MFs, the cells collected after peritoneal lavage were incubated with Tim4 (PE) Ab and purified with anti-PE MicroBeads (Miltenyi Biotec).
Generation of ACs To generate ACs, thymocytes isolated from 4–6-wk-old C57BL/6 mice were incubated in RPMI 1640, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin, in the presence of 1 mM dexamethasone (Sigma-Aldrich). After 6 h, the thymocytes were washed several times with PBS, spun through a FCS cushion to eliminate the dexamethasone, and resuspended in RPMI 1640 containing 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. The frequency of ACs was checked by annexin V/PI staining. MFs were incubated with ACs at a 1:5 ratio at 37˚C for the indicated times.
The Journal of Immunology
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Phospholipid preparation 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl3-sn-phosphoserine were purchased from Avanti Polar Lipids. Phospholipids were dissolved in chloroform and stored at 280˚C until their use. For each experiment, the indicated lipids were evaporated under a stream of argon and resuspended in culture medium by vigorous vortexing for $30 s. For the generation of phosphatidylserine-containing liposomes, 1-palmitoyl2-arachidonoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-3-sn-phosphoserine were mixed at a 4:1 ratio.
age. Mice were sacrificed 4 mo later. Spontaneous autoimmune phenomena were assessed in mice without any stimulus or challenge at 26 wk of age. For detection of autoantibodies, microtiter plates were coated overnight with 50 mg/ml histone of calf thymus (Roche Diagnostics) dissolved in ethanol. For detection of anti-dsDNA Abs, plates were coated with 20 mg/ml poly-L-lysine before coating with 20 mg/ml calf thymus DNA (both from Sigma-Aldrich). Sera were added at a 1:100 dilution in 1% BSA/PBS for 1 h at room temperature. Bound IgG was detected with an HRP-conjugated goat anti-mouse IgG (Dianova).
In vitro phagocytosis assay
Real-time PCR
A mixture of WT resident and thioglycollate-elicited peritoneal MFs was isolated 5 d after i.p. injection of 1 ml 3% Brewer’s thioglycollate, plated on coverslips in 24-well plates, and incubated with CFSE-labeled apoptotic thymocytes, at a 1:5 ratio, in 10% FCS in RPMI 1640 for 30 min at 37˚C. Unbound cells were washed away with ice-cold PBS, and the MFs were stained as indicated and mounted on glass slides. Microscopy was performed with an Eclipse-80i microscope and a monochromatic camera (DSQi1MC; both from Nikon). Photomicrographs are shown with indicated pseudocolors produced by NIS elements software BR3.0 (Nikon).
In brief, total RNA was isolated from cells using peqGOLD TriFast (PEQLAB). A total of 700 ng total RNA was reverse transcribed with human leukemia virus reverse transcriptase using the Gene Amp RNA PCR kit (Applied Biosystems) and oligo(dT) primers. Total RNA from the spleen was isolated by the same procedure, but DNA digestion took place prior to transcribing the RNA (DNase I, #EN0521; Fermentas). mRNA levels were quantified using a LightCycler instrument and SYBR Green I kit (Roche Diagnostics). The gene expression values were normalized to b-actin expression. The sequence of the primers can be found in Supplemental Table I.
In vivo phagocytosis assay
Western blotting
Five days after i.p. injection of 1 ml 3% Brewer’s thioglycollate, 15 3 10 CFSE-labeled apoptotic thymocytes were injected i.p. into WT mice. After 30 min, peritoneal lavage was performed, and the cells were stained for Tim4 (PE), CD115 (allophycocyanin), and F4/80 (Pacific Blue) and analyzed with a Gallios cytofluorometer (Beckman Coulter) and FlowJo software. 6
In vivo AC uptake A total of 5 3 107 CFSE-labeled apoptotic thymocytes was injected i.v. into 10–12-wk-old WT and NR4A12/2 mice. The mice were sacrificed after 18 h. The spleens were collected, and one part was embedded in OCT Tissue-Tek compound, snap-frozen, and stored at 280˚C; the other part was used for RNA analysis.
Pristane model Autoimmunity was induced in NR4a12/2 mice and C57BL/6 mice with a single i.p. injection of 0.5 ml pristane oil (Sigma-Aldrich) at 10 wk of
FIGURE 1. Apoptotic cells are preferentially engulfed by resident MFs. (A) Five days after injection of thioglycollate, 15 3 106 CFSElabeled apoptotic thymocytes were injected into the inflamed peritoneum, and flow cytometric analysis was performed to determine the uptake of apoptotic cells by Tim4+ resident MFs and Tim42 inflammatory MFs. After exclusion of the doublets, the cells were gated on CD115+ cells, and the intensity of CFSE was determined in both populations of MFs. (B) Immunofluorescence microscopy analysis and immunofluorescence-based quantification of the phagocytosis of ACs (green) by MFs isolated 5 d after initiation of thioglycollate-induced peritonitis. MFs were stained for TIM4 (red) and CD68 (blue) to differentiate between TIM4+ resident peritoneal MFs and TIM42 inflammatory monocyte-derived MFs. Scale bar, 10 mm. (C) Immunofluorescence microscopy analysis of the phagocytosis of ACs (green) by a pure population of resident peritoneal MFs. (D) After induction of thioglycollateinduced peritonitis, Tim4+ MFs and Tim42 MFs were separated by MicroBeads, as described in Materials and Methods, and relative mRNA expression of the indicated receptors was determined by real-time PCR in unstimulated resident MFs and Tim42 inflammatory peritoneal MFs. The data shown are representative of at least three independent experiments. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.01.
After lysis of the cells, the protein extracts were separated on 10% SDS– polyacrylamide gel, transferred onto a polyvinylidene difluoride membrane, and immunoblotted with the following Abs: phospho–NF-kB– p65 (#3033; Cell Signaling) and b-actin (clone AC-74; Sigma-Aldrich). Quantitative analyses were performed with Photoshop CS5 software, and the results were normalized to the controls (= 1).
Reagents Murine IL-12p40, TNF-a, IL-6, and CXCL1 were measured by specific ELISA kits (R&D Systems). LPS was purchased from Sigma-Aldrich (#L-2630; Escherichia coli 0111:B4). All inhibitors were used at 10 mM. The PI3K inhibitor (wortmannin) was purchased from Sigma-Aldrich, the PKC inhibitor (Bisindolylmaleimide I, Hydrochloride) was purchased from Cell Signaling, the PKA inhibitor (KT 5720) was purchased from Santa Cruz, and the NF-kB activation inhibitor, the p38 inhibitor (SB203580), and the p44/p42 inhibitor (PD98059) were purchased from Merck Millipore.
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Latex beads (size 1 mm, 0.01%) and CFSE were purchased from SigmaAldrich, and cycloheximide (CHX) was purchased from Merck Millipore. The polyclonal anti-F4/80 (labeled with either PE or Pacific Blue), antiTIM4 (PE), anti-CD68 (allophycocyanin), and anti-CD115 (allophycocyanin) Abs were purchased from BioLegend, and anti-SIGNR1 (allophycocyanin) was from eBioscience.
Results
Statistical analysis Data are mean 6 SEM. Group mean values were compared using the two-tailed Student t test. The data shown are representative of at least three experiments producing similar results.
FIGURE 2. Clearance of ACs induces expression of the Nr4a family of NRs. (A) ELISA-based analysis of the cytokine profile of resident MFs after preincubation (1 h) with ACs (ratio 1:5) and stimulation (overnight) with LPS (100 ng/ml). (B) Resident peritoneal MFs were incubated for 30 min with 10 mg/ml of CHX and then incubated with ACs (ratio 1:5) for 1 h before being stimulated with 100 ng/ml of LPS for 4 h. The relative mRNA expression of IL-12p40 was analyzed by real-time PCR. Resident peritoneal MFs were incubated with ACs (ratio 1:5) for the indicated times, and relative mRNA expression of the indicated early response genes (C) and Nr4a family members (D) was determined by real-time PCR. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.01.
Tissue-resident MFs perform the noninflammatory clearance of ACs Recent studies revealed a surprising dichotomy among the various MF subsets encountered in vivo. In addition to classical monocytederived MFs, which are recruited during inflammation, a large group of tissue-resident MFs, such as Kupffer cells, splenic MFs, alveolar MFs, and resident peritoneal MFs settle the tissues during embryogenesis. Notably, such resident MFs sustain their numbers via local proliferation throughout adulthood independent from monocyte-derived precursors (15–17). However, little is known about the differential function and specific properties of inflammatory (monocyte-derived) and resident MFs. Our previous studies indicate that clearance of ACs by tissueresident MFs maintains self-tolerance, whereas uptake of ACs by inflammatory monocytes and monocyte-derived MFs eventually results in an immune response to AC-derived self-Ags (18). To better understand the underlying molecular mechanisms, we studied the phagocytosis of ACs by inflammatory and resident MFs of the peritoneal cavity. By using Tim4 as a specific marker for resident peritoneal MFs (19, 20), we were able to distinguish the two populations of MFs during flow cytometry and via immunofluorescence (Fig. 1A, 1B). In accordance with our previous data, we observed that the phagocytosis of CFSE-labeled ACs was enhanced in the population of resident MFs, whereas inflammatory MFs bound and ingested ACs to a lesser extent (Fig. 1A, 1B). Even within an inflammatory infiltrate, where monocyte-derived inflammatory MFs were the prevalent cell type, the remaining resident MFs
FIGURE 3. ACs induce expression of Nr4a1 via the p38MAPK and ERK 1/2 (p44/p42) pathway in a PS-dependent manner. (A) Resident peritoneal MFs were incubated (1 h) with either ACs (1:5) or latex beads (2%) before Nr4a1 mRNA expression was determined. (B) Analysis of Nr4a1 mRNA expression after stimulation (1 h) of resident MFs with ACs or ACs coupled with annexin V (AC+AxV). (C) Resident MFs were treated with PS-containing phospholipid liposomes (PS-lipo.) for the indicated times, and relative mRNA expression of Nr4a1 was determined by real-time PCR. (D) Resident peritoneal MFs were preincubated with the indicated inhibitors (inh.) for 30 min before addition of ACs. The expression of Nr4a1 was analyzed after an additional hour by real-time PCR. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.01.
The Journal of Immunology exerted AC removal that outpaced the clearance performed by inflammatory MFs. Accordingly, uptake of ACs by a pure population of resident MFs was highly efficient and homogenous (Fig. 1C). mRNA expression analysis of purified populations of resident and inflammatory MFs showed that the enhanced capacity of resident MFs to ingest ACs was paralleled by a differential expression of certain receptors for ACs (Fig. 1D, Supplemental Fig. 1). These data supported a concept whereby ACs are preferentially cleared by resident, but not monocyte-derived, MFs. However, previous studies addressing the clearance of ACs focused on the phagocytosis by monocyte-derived MFs or MF cell lines. The molecular details of the phagocytosis of ACs by tissue-resident MFs have received little attention. Phagocytosis of ACs by tissue-resident MFs results in the induction of a unique pattern of immediate early genes and the differential blockade of cytokine expression We subsequently sought to gain mechanistic insight into the effects that ACs exert on this specialized subset of MFs. Resident MFs responded with a differential inhibition of the LPS-induced cytokine release after phagocytosis of ACs, and we observed an almost complete inhibition of the expression of distinct proinflammatory cytokines, such as IL-12p40, whereas expression of other cytokines, such as CXCL1, was not affected by the uptake of
4855 ACs (Fig. 2A). Because these AC-induced changes in the cytokine response of MFs were observed with a delay of several hours, we hypothesized that the regulation of cytokine expression in this type of MF relied on earlier transcriptional events that are directly initiated upon the ingestion of ACs. In accordance, CHX-induced inhibition of ribosomal protein translation resulted in abrogation of the AC-induced block of IL-12 expression (Fig. 2B), suggesting that ACs induced the rapid synthesis of proteins that negatively regulated inflammatory cytokine production by MFs. To screen for potential candidates, we subsequently performed an analysis of the MF’s immediate early gene expression, which showed vigorous transcriptional changes in response to the ingestion of ACs (Fig. 2C). These experiments resulted in the identification of the Nr4a family of NRs, and in particular, Nr4a1, as one of the most robustly induced set of genes (Fig. 2D, Supplemental Fig. 2). Because Nr4a1 was recently implicated as a key factor regulating the inflammatory response (8), we subsequently addressed its role during the effects that ACs exerted on resident MFs. ACs induce Nr4a1 in a phosphatidylserine-dependent manner via activation of p38MAPK and MAP/ERK Notably, we did not observe expression of Nr4a1 in response to the phagocytosis of other particles, such as latex beads (Fig. 3A), indicating that signals initiated by the specific binding of ACs, and
FIGURE 4. Nr4a1 contributes to the anti-inflammatory effects of ACs. (A) Real-time PCR of Nr4a1 mRNA expression in resident MFs after preincubation (2 h) with ACs and treatment (1 h) with 100 ng/ml of LPS. (B) ELISA-based analysis of the IL-12p40 and CXCL1 expression of resident Nr4a1+/+ and Nr4a12/2 peritoneal MFs after preincubation (1 h) with ACs (ratio 1:5) and overnight treatment with 100 ng/ml of LPS. (C) ELISA-based analysis of the IL-12p40 and CXCL1 expression of resident Nr4a1+/+ and Nr4a12/2 peritoneal MFs after preincubation (1 h) with PS-liposomes (PS-lipo.) and overnight treatment with 100 ng/ml of LPS. Western blot analysis of phosphorylation of the NF-kB subunit p65 (Ser536) in Nr4a1+/+ and Nr4a12/2 peritoneal MFs after preincubation (2 h) with ACs (D) or PS-containing liposomes (E) and stimulation with 100 ng/ml LPS for the indicated times (D) or for 5 min (E). Data are mean 6 SEM. *p , 0.05.
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not the process of phagocytosis per se, triggered the expression of this NR in resident MFs. Exposure of the phospholipid phosphatidylserine (PS) on the surface of cells undergoing apoptosis was shown to serve as a major “eat me” signal for phagocytes (21), and our previously obtained data showed that resident MFs were well equipped with PS-specific receptors for ACs, such as Tim4. To address a potential involvement of PS exposure during the effects exerted by ACs, we subsequently performed phagocytosis assays in the presence of annexin V, a protein that masks the PS moieties on the surface of ACs. Addition of annexin V blocked the AC-induced expression of Nr4a1 in resident MFs (Fig. 3B), whereas the addition of PS-containing liposomes induced the expression of Nr4a1, also in the absence of ACs (Fig. 3C). Together, these data suggested a key role for PS recognition by resident MFs during the AC-mediated induction of Nr4a1 expression in this MF subpopulation. To further address the underlying signaling pathways, we applied different inhibitors of the p38MAPK, MAP/ERK 1/2 (p44/ p42), protein kinase C, protein kinase A, and NF-kB pathway in our assays. We observed a significant inhibition of AC-induced NR4a1 expression after blockade of both the p38MAPK and the MAP/ERK kinase 1/2 pathways, whereas the other inhibitors did not interfere with the effects exerted by ACs, or they even slightly increased Nr4a1 expression (Fig. 3D).
tion of ACs resulted in attenuated splenic mRNA expression of the gene encoding for IL-12 subunit p40 (Il12b) in WT animals, whereas expression of Il12b increased in the spleens of Nr4a12/2 mice in response to ACs (Fig. 5C). In contrast, Nr4a1 expression did not significantly impact on the expression of the gene encoding for CXCL1 (Fig. 3C). To address a potential contribution of Nr4a1 to the maintenance of tolerance to AC-derived Ags in vivo, we used the pristane model of experimental murine lupus, in which i.p. injection of pristane oil results in the induction of a chronic and sterile peritonitis that is accompanied by overwhelming cell death within the peritoneal cavity. Together, these events trigger a break of tolerance to ACderived nuclear Ags and result in the production of autoantibodies against dsDNA and histones (25). Injection of pristane oil resulted in the occurrence of autoantibodies against nuclear Ags in Nr4a12/2 mice, whereas their WT littermates developed significantly lower titers of autoantibodies (Fig. 5D), confirming a critical role for Nr4a1 in the maintenance of immunologic tolerance to AC-derived self-Ags.
Nr4a1 supports the maintenance of an anti-inflammatory milieu during the clearance of ACs Nr4a family members act as crucial regulators of the inflammatory response (8, 10, 22). Consequently, we sought to determine a potential role for Nr4a1 during the maintenance of an antiinflammatory cytokine milieu in response to the clearance of ACs by resident MFs. In contrast to the expression of IL-12, expression of Nr4a1 was synergistically induced by ACs and LPS (Fig. 4A). Notably, ACs exhibited reduced anti-inflammatory effects in the absence of this NR (Fig. 4B), which resulted in the restoration of LPS-induced IL-12p40 production in Nr4a12/2 MFs after phagocytosis of ACs. These NR4a1-mediated effects were specific for cytokines such as IL-12p40, because neither phagocytosis of ACs nor the presence of Nr4a1 affected the expression of other factors, such as CXCL1. Our previous data indicated a key role for AC-derived PS during the regulation of NR4a1. Accordingly, we observed that PS-containing liposomes blocked production of IL-12p40 but not of CXCL1 (Fig. 4C). Again these anti-inflammatory effects involved Nr4a1, because we observed reduced inhibitory effects of PS-containing liposomes in Nr4a12/2 resident MFs (Fig. 4C). The NF-kB pathway acts as a master regulator of the inflammatory response in MFs (23), and uptake of ACs was reported to interfere with NF-kB activity in various cell types (24). Notably, analysis of the NF-kB–signaling cascade revealed a block of the LPS-induced phosphorylation of NF-kB subunit p65 at serine 536 in resident MFs ingesting either ACs or PS-containing liposomes (Fig. 4D, 4E). Notably, these effects were abrogated in Nr4a12/2 resident MFs, indicating an AC-induced and Nr4a1-mediated block of the phosphorylation of p65 (Fig. 4D, 4E). NR4a1 mediates the anti-inflammatory effects of ACs in vivo and contributes to the maintenance of self-tolerance To evaluate the contribution of Nr4a1 to the anti-inflammatory effects of ACs in vivo, we subsequently injected ACs i.v. into WT and Nr4a12/2 mice. ACs were predominantly scavenged by Tim4+SIGNR1+ marginal zone MFs within the spleen (Fig. 5A), which resemble another type of resident MFs (17). Again we observed a rapid induction of Nr4a1 and (to a lesser extent) Nr4a2 expression (Fig. 5B). In accordance with our in vitro data, injec-
Discussion AC death constantly occurs during regular tissue turnover and, in particular, during the course of an inflammatory response. Because ACs represent a major source of autoantigens, their rapid and nonimmunogenic removal by MFs is essential for the maintenance of self-tolerance. Notably, ACs themselves exert direct anti-inflammatory effects, which additionally increase the threshold for an immune response against AC-derived Ags. Our data indicate that mainly tissue-resident, and not monocytederived, MFs perform the clearance of ACs. These findings are in accordance with previous studies that highlighted the importance of specialized and tolerogenic MF subsets, such as marginal zone
FIGURE 5. Nr4a1 promotes the noninflammatory clearance of ACs and contributes to the maintenance of self-tolerance. After i.v. injection of 50 3 106 CFSE-labeled ACs into WT mice, immunofluorescence and mRNA expression analysis were performed to determine the localization of ACs (A), as well as the expression of Nr4a family members (B). (C) After i.v. injection of 50 3 106 CFSE-labeled ACs into Nr4a1+/+ and Nr4a12/2 mice, spleens were harvested, and an mRNA expression analysis of the indicated genes was performed by real-time PCR. Data shown are representative of two independent experiments (n = 3 or 4). (D) Serum analysis of the indicated autoantibodies in Nr4a1+/+ and Nr4a12/2 mice 16 wk after i.p. injection of 0.5 ml of pristane oil. Data are mean 6 SEM. Scale bars, 100 mm. *p , 0.05, ***p , 0.01.
The Journal of Immunology MFs, during the noninflammatory clearance of dying cells (26, 27). Upon binding of ACs, resident MFs rapidly induced the expression of multiple immediate early genes, including the Nr4a family of NRs. Recognition of PS on the surface of ACs was a key trigger for these AC-induced transcriptional changes, and PSinduced Nr4a1 critically contributed to the immune-modulatory properties of ACs. Because PS exposure is a universal feature of AC death, it is likely that similar events occur during the uptake of apoptotic thymocytes, as well as during the clearance of other cell types, such as apoptotic neutrophils. Thus, our data support a scenario in which PS exposure on ACs serves as a crucial “eat me” signal and likewise triggers anti-inflammatory signaling cascades within the MF that alter its phenotype (28, 29). Indeed, exposure of PS is essential for the anti-inflammatory effects of ACs (30, 31); and during the last several years several PS-binding receptors, such as Tim4 and MerTK, were identified and shown to mediate the nonimmunogenic clearance of ACs (19, 20, 24, 32–37). On a mechanistic level, Nr4a1 interfered with the LPS-induced phosphorylation of NF-kB subunit p65, as well as with the expression of IL-12. Nr4a family members were shown to attenuate overwhelming inflammation by recruiting transcriptional corepressors to distinct NF-kB target genes (21). Such a scenario might also explain the Nr4a1-induced blockade of p65 phosphorylation (9) and IL-12 expression. Although deletion of NR4a1 alone significantly interfered with the anti-inflammatory effects of ACs, it was not sufficient to completely abrogate them. This fact is most probably due to a considerable degree of redundancy in the function of NR4a1-3, as well as to the potential contribution of other factors that were reported to modulate the cytokine response of MFs in reaction to the uptake of ACs (38). Notably, ACs and LPS synergistically induced the expression of Nr4a1, indicating that this NR integrates both pro- and anti-inflammatory signals on the transcriptional level. Thereby, Nr4a1 seems to maintain a certain inhibitory threshold, blocking the initiation of an immune response when cell death and inflammation come together. In the pristane model of experimental murine lupus, injection of pristane oil triggers a chronic inflammatory response that is paralleled by an increased rate of apoptotic and necrotic cell death (39). Although WT mice are able to cope with this situation, alterations in the noninflammatory clearance of ACs, such as the deletion of Tim4 (40) or absence of Nr4a1, eventually result in the break of self-tolerance to AC-derived Ags. Also, other NRs, such as peroxisome proliferator-activated receptors and liver X receptors, were shown to act as transcriptional sensors for dying cells and to subsequently orchestrate their noninflammatory clearance (41, 42). Together with our current data, these findings highlight a key role for NRs as sensors of cell death and coordinators of tissue homeostasis that maintain immune tolerance to “dying self.” Therefore, NRs might impose interesting targets in the therapy of diseases linked to a break of self-tolerance, such as systemic lupus erythematosus or rheumatoid arthritis.
Acknowledgments We thank C. Stoll and I. Schmidt for excellent technical assistance.
Disclosures The authors have no financial conflicts of interest.
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