Introduction: Macrophages abundantly express liver X receptors (LXRs), which are ligand-dependent transcription factors and sensors of several cholesterol metabolites. In response to agonists, LXRs promote the expression of key lipid homeostasis regulators. Cross talk between LXRs and inflammatory signals exists in a cell type- and gene-specific manner. A common feature in the macrophage response to inflammatory mediators is the induction of CCAAT/enhancer-binding protein beta (C/EBPβ), a master transcriptional regulator and lineage-determining transcription factor in monocytes/macrophages. Methods: Quantitative real-time PCR in control and C/EBPβ-deficient macrophages was used to explore the role of C/EBPβ in the cross talk between inflammatory mediators and the macrophage response to pharmacological LXR activation. The functional interaction between C/EBPβ and LXRs on selected genomic regions was further characterized by chromatin-immunoprecipitation (ChIP) and gene reporter studies. Results: Whereas inflammatory signaling repressed several LXR-regulated genes involved in lipid metabolism, these effects were conserved after deletion of C/EBPβ. In contrast, inflammatory mediators and LXRs synergistically induced the expression of the multifunctional protein CD38 in a C/EBPβ-dependent manner. C/EBPβ and LXRs bound to several regions with enhancer activity upstream and within the mouse Cd38 gene and their functional cooperation in macrophages required intact binding sites for LXR and C/EBPβ. Conclusion: This study reveals positive cross talk between C/EBPβ and LXRs during the macrophage inflammatory response, which selectively impacts CD38 expression.

Macrophages are innate immune cells that play key roles in the host defense against insults of diverse origin, but they are also integral tissue components involved in the regulation of metabolism and homeostasis [1]. The plethora of activities performed by macrophages is, in part, a consequence of their great phenotypical plasticity in adapting to signals from the microenvironment [2]. In this regard, the integration of environmental signals impacts macrophage metabolism, which in turn modulates the characteristics of the response.

Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors, which play diverse roles in the cross talk between inflammatory pathways and lipid metabolism in macrophages (reviewed in [3]). LXRs are sensors of natural cholesterol metabolites, but can also be pharmacologically activated by highly specific synthetic agonists, such as TO-901317 (T1317) and GW3965. Two LXR subtypes exist, namely LXRα and LXRβ, encoded by separate genes. While LXRβ is ubiquitously expressed in the body, LXRα predominates in tissues and cells with high metabolic activity, including macrophages (reviewed in [4]).

LXRs form heterodimers with another nuclear receptor, the retinoid X receptor (RXR). LXR-RXR heterodimers bind to LXR response elements (LXREs) on DNA and interact with corepressor complexes to repress gene transcription in the absence of an agonistic ligand [5, 6]. Activation of LXR-RXR heterodimers with an LXR agonist promotes the exchange of corepressors for coactivators to induce gene transcription (reviewed in [7]). LXRs control the expression of key regulators of cholesterol, fatty acid, and phospholipid homeostasis (reviewed in [3]), including the sterol transporters ATP binding cassette A1 (ABCA1) and G1 (ABCG1), which promote cellular cholesterol efflux [8, 9], several apolipoproteins (Apo), such as ApoC1, involved in lipid transport [10], and sterol regulatory element-binding protein 1 (SREBP1c; encoded by the gene Srebf1), which activates the transcription of lipogenic genes [11, 12].

In myeloid cells, LXRs also control the expression of molecules involved in immune responses, including the macrophage apoptosis inhibitory factor CD5L [13, 14], the receptor MER tyrosine kinase (MERTK) with important roles in the phagocytosis of apoptotic bodies [15], and the multifunctional protein CD38 [16, 17].

CD38 is expressed both as an integral membrane protein and as an extracellular soluble form and is a therapeutic target in several types of cancer [18]. Membrane-bound CD38 has several functions as a receptor [19] or co-receptor in association with other immune protein complexes (reviewed in [20]), and as an enzyme (reviewed in [18]). The enzymatic activities of CD38 result in large consumption of nicotinamide adenine dinucleotide (NAD) to generate calcium-mobilizing second messengers, for which reason CD38 is considered to be the major regulator of NAD levels in mammalian tissues [21]. The loss of functional CD38 expression and/or activity is associated with impaired immune responses and metabolic and behavioral alterations [18, 22‒24].

Pieces of evidence support reciprocal negative cross talk between pharmacologically activated LXRs and inflammatory signals. LXR agonists antagonize proinflammatory gene expression through a combination of mechanisms, including transrepression [25] and the modulation of genes involved in cholesterol metabolism [26, 27]. In turn, proinflammatory signaling represses the induction of several LXR target genes involved in lipid homeostasis [28, 29]. However, in contrast with these mutually opposing effects, LXRs and several inflammatory mediators, namely tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and lipopolysaccharide (LPS), cooperated to synergistically increase the expression and NADase activity of CD38 in murine macrophages [17]. Cooperation with inflammatory signaling was observed when either synthetic (T1317 or GW3965) or natural (25-hydroxycholesterol) LXR agonists were used [17].

Despite the usage of different signal transduction modules, a common feature in the macrophage response to these inflammatory mediators is the induction of CCAAT/enhancer-binding protein beta (C/EBPβ), a transcription factor that acts as a master regulator of gene expression in monocytes/macrophages [30]. Members of the C/EBP family are characterized by a highly conserved basic leucine zipper domain by which they dimerize and bind to palindromic α-helical recognition sequences in the DNA, and an effector domain that mediates transactivation or repression [31]. After protein synthesis, C/EBPβ rapidly translocates to the nucleus to regulate transcription by binding to DNA either as a homodimer or as a heterodimer with other transcription factors [32].

C/EBPβ is also considered a lineage-determining transcription factor (LDTF) [33, 34], important for priming cis-regulatory elements required for macrophage identity [35]. Accumulated evidence supports a model in which combinations of LDTFs collaborate on selected enhancers providing cell type-specific sites of open chromatin that facilitate the binding of signal-dependent transcription factors [36]. This model was initially exemplified by the requirement of the LDTF PU.1 for the induction of LXR- and Toll-like receptor (TLR)-4-dependent gene expression in macrophages [35]. Based on the association of high C/EBPβ expression with the proinflammatory response of macrophages [37, 38], in this work we have explored the relevance of this transcription factor in the cross talk between inflammatory signals and the LXR transcriptional program in macrophages. Activation of macrophages with inflammatory mediators resulted in changes in the expression of LXR and RXR subtypes as well as in the transcriptional response to an LXR agonist. Whereas C/EBPβ was dispensable for most of these changes, the cooperative induction of CD38 by LXRs and inflammatory signaling fully required C/EBPβ expression. The cooperative effect between the LXR pathway and C/EBPβ was further supported by their binding profile in the proximity of the gene encoding for mouse Cd38 and by functional collaboration in gene-reporter studies.

Reagents

The synthetic and high-affinity LXR agonists T1317 and GW3965 were purchased from Cayman Europe and Tocris, respectively. The RXR agonist LG268 was synthetized as described [17, 39]. Recombinant murine IFNγ or TNFα was purchased from PeproTech. LPS from Escherichia coli 0127:B8 was obtained from Sigma-Aldrich.

Animals

C57BL/6 mice were purchased from Envigo and raised as a colony in our animal facility. LXR-deficient mice were initially donated by Dr. David Mangelsdorf (UT Southwestern Medical Center, Dallas, TX, USA) and backcrossed into C57BL/6 background for more than 10 generations. Mice with myeloid C/EBPβ deficiency were generated by crossing transgenic mice expressing Cre-recombinase under the lysozyme M (LysM) promoter (B6.129P2-Lyz2tm1(cre)Ifo/J 004781, Jackson Laboratories) with mice carrying a C/EBPβ gene flanked by LoxP sites [40] kindly donated by Dr. Esta Sterneck (National Cancer Institute, Frederick, MD, USA). These mice were in C57BL/6 background and are referred to as LysMCre-C/EBPβfl/fl hereafter. Control littermates were transgenic for C/EBPβfl/fl but negative for the Cre-recombinase [38]. CD38-deficient mice correspond to JAX stock #003727 [41] backcrossed in the C57BL/6 background for more than 10 generations.

Cells

Bone marrow-derived macrophages (BMDMs) were obtained from 6- to 10-week-old mice as described [42]. Briefly, bone marrow precursors were differentiated to macrophages during 7 days in DMEM supplemented with 20% heat-inactivated fetal bovine serum (Sigma-Aldrich) and 30% L929-conditioned media as a source of macrophage-colony-stimulating factor.

RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR Analysis

Total RNA was extracted from cells using Trizol (Invitrogen) as recommended by the manufacturer. For cDNA synthesis, 1 μg of RNA was reverse transcribed with M-MLV Reverse transcriptase RNase H Minus, Point Mutant, oligo(dT)15, and PCR nucleotide mix (Promega). Quantitative real-time PCR was carried out using the Power SYBR Green Reagent Kit (Applied Biosystems). Specific primers used in this study are shown in online supplemental Table 1 (for all online suppl. material, see https://doi.org/10.1159/000543274). The data are presented as mRNA levels relative to ribosomal L14 expression.

Protein Extraction and Western Blot Analysis

The cells were placed on ice, washed with PBS, and lysed in RIPA lysis solution (50 mm Tris-HCl pH 7.4, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 150 mm NaCl, 2 mM EDTA, 50 mm NaF) supplemented with protease inhibitors (1 mm phenylmethylsulfonyl fluoride and a protease inhibitor cocktail; Santa Cruz Biotechnology). Insoluble material was removed by centrifugation at 14,000× g for 15 min at 4°C. Cell lysates (20 µg) were boiled for 5 min at 95°C in Laemmli SDS loading buffer. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Immobilon-FL). The membranes were blocked in Odyssey blocking buffer (Li-Cor) diluted 1:1 with TBS-0.05% Tween 20 (TBS-T) and later incubated with a rabbit polyclonal anti-C/EBPβ antibody (Santa Cruz Biotechnology, #sc-150) or a goat polyclonal anti-CD38 antibody (Santa Cruz Biotechnology, #sc-7049). Mouse monoclonal anti-α-tubulin (Life Technologies, #13-8000) or rabbit polyclonal anti-β-tubulin (Cell Signaling, #2146) antibodies were used to monitor differences in protein loading. After incubation with primary antibodies, the membranes were washed three times in TBS-T and incubated for 1 h with peroxidase-conjugated secondary antibodies. After three washes with TBS-T, enhanced chemiluminescence detection was performed (GE Healthcare) and the membranes were exposed to X-ray films (Fujifilm) (in the case of CD38 detection) or quantified in an Odyssey Fc Imaging System (Li-Cor) with the Image Studio™ Lite software (Li-Cor) (to study relative C/EBPβ isoform expression).

Enzyme-Linked Immunosorbent Assay

Supernatants were collected after treating macrophages with LPS (100 ng/mL) for different periods of time. The levels of secreted IL-6 and IL-12 were measured using specific ELISA kits (PeproTech, #900-K50 and #900-K97, respectively) and following the manufacturer’s recommendations.

Analysis of RNA Sequencing Data

Public RNA-sequencing (RNA-Seq) data were obtained from the GEO NCBI database with the accession number GSE90046 [38, 43] (online suppl. Table 2). RNA readings were aligned to the mm10 version of mouse genome with Rsubread [44]. The RNA-Seq count matrix was analyzed using DESeq2 R package, a method based on the negative binomial distribution with variance and mean linked by local regression [45]. Normalized whole read counts were used for the evaluation of changes in the expression of several genes of interest in primary microglia from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice stimulated with LPS or vehicle [38, 43]. For data visualization, a heatmap was produced with Heatmapper (Wishart Research Group, University of Alberta, Canada).

Identification of Enhancer Regions

Public chromatin immunoprecipitation-sequencing (ChIP-Seq) datasets were obtained from the GEO NCBI database (online suppl. Table 3) as indicated below. ChIP-Seq data were obtained from experimental approaches performed in BMDMs for various transcription factors and histone modifications. In more detail, ChIP-Seq data for CEBPβ were obtained from the accession number GSE109965 [46], using BMDMs from C57BL/6J mice, either left untreated or stimulated with Kdo2-Lipid A for 1 h. ChIP-Seq data for RXR correspond to BMDMs, either control or treated with GW3965 for 1 h, from the accession number SRP019970 [47]. ChIP-Seq data for acetylated histone H3 on lysine 27 (H3K27ac) carried out in BMDMs, either control or stimulated with LPS for 2, 4, or 24 h, were obtained from accession numbers GSE56123 [48] and GSE38377 [49]. ChIP-Seq for LXRα was performed using a dual LXRα/β antibody [50] in LXRβ-deficient BMDMs, either left untreated or treated with GW3965 in combination or not with LPS for 24 h. These data are available under accession numbers GSE200922 [51] and GSE275506. All sequencing data were mapped to the mm10 assembly of the mouse genome using Bowtie2. The data were then analyzed using HOMER [35], with each sequencing experiment normalized to 107 uniquely mapped tags. Sequencing experiments were visualized with IGV genome browser. Regions of interest with pronounced H3K27ac marks were scanned for LXRE and C/EBPβ binding motifs using public databases and motif matrices through R [52] and Bioconductor packages, including MotifDb [53] and Universalmotif [54].

Site-Directed Mutagenesis

We had previously cloned a 613-bp genomic region (using primers 5′ACCTGCTGGACTGTGTCCTT3′ and 5′CCTTTGAGGGGTCCTTTCTC3′) containing the major part of the mouse CD38 enhancer region R2 within a pGL3 promoter-luciferase plasmid (Promega) [17], hereafter named pGL3-R2. A modified version of that plasmid was also available containing four site mutations in an LXRE within R2 [17]. In the current study, specific mutations were introduced into the C/EBPβ binding site identified within this region using a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). All the plasmids were sequenced to confirm correct cloning and mutations. Plasmid DNA was prepared using Maxi-prep columns (JetStar 2.0 Kit from Genomed).

Reporter Activity Assays

Raw264.7 macrophages or COS-7 kidney cells were plated in 12-well plates (2 × 105 cells/well), and 24 h later, the cells were co-transfected with 500 ng of reporter plasmid pGL3-R2, 300 ng of either pcDNA3-LXRα or pcDNA3-LXRβ, 300 ng of pcDNA3-RXRα, and/or 300 ng of pMSV-C/EBPβ. Alternatively, variants of pGL3-R2 containing four point mutations in an LXRE [17] or in a C/EBPb binding site were used in some experiments. Empty vector (pcDNA3 and/or pMSV) was used in control transfections. To control transfection efficiency, all transfections included 100 ng of a Renilla luciferase-expressing plasmid (pRL-TK). Transfections were carried out using SuperFect Transfection Reagent (Qiagen) following the manufacturer's instructions. Luciferase activity was assessed using the Firefly & Renilla Luciferase Single Tube Assay Kit (Biotium) in an Infinite M200 luminometer (Tecan). Firefly luciferase activity values were normalized to those of Renilla luciferase. In each experiment, all experimental conditions were evaluated in duplicates or triplicates.

Chromatin-Immunoprecipitation Assays

Cell fixation and cross‐linking were performed in two steps. First, 20 × 106 cells were cross‐linked with 2 µm disuccinimidyl glutarate (Thermo Fisher Scientific) diluted in PBS for 30 min. Then, the cells were washed with PBS and incubated with 1% formaldehyde (Merck) in PBS for 10 min. Cross‐linking was quenched with 200 mm glycine (Merck) for 10 min. Chromatin was extracted with a two‐step lysis method. First, the cells were swollen with hypotonic buffer (50 mm Tris-HCl pH 8, 85 mm KCl, 0.5% IGEPAL, and protease inhibitors) and then incubated with lysis buffer (50 mm Tris-HCl, pH 8, 10 mm EDTA, 1% SDS, and protease inhibitors). Chromatin was sonicated with a Sonopuls sonicator (Bandelin), yielding 300–1,000 bp fragments. A fraction (10%) of the total volume was kept as input control. Immunoprecipitation was performed with 2 µg of rabbit anti-LXRα/β IgG [55], 1:500 dilution of rabbit anti-RXRβ IgG (Invitrogen, # PA1-815), or 1 µg rabbit anti-C/EBPβ IgG (Santa Cruz Biotechnology, # sc-150) in 1 mL dilution buffer (10 mm Tris-HCl, pH 8, 2 mm EDTA, 1% Triton X‐100, 150 mm NaCl, 5% glycerol, and protease inhibitors) during 15 h at 4°C. Antibody‐bound complexes were recovered with protein G Dynabeads (Life Technologies). The complexes were sequentially washed once with buffer I (20 mm Tris-HCl, pH 8, 2 mm EDTA, 1% Triton X‐100, 0.1% SDS, 150 mm NaCl), buffer II (20 mm Tris-HCl, pH 8, 2 mm EDTA, 1% Triton X‐100, 0.1% SDS, 500 mm NaCl), and buffer III (10 mm Tris-HCl pH 8, 1% sodium deoxycholate, 1 mm EDTA, 1% IGEPAL, 250 mm LiCl), and twice with TE buffer (10 mm Tris-HCl pH 8, 1 mm EDTA). Reverse cross‐linking of protein‐DNA fragments was performed by incubating the samples in reverse cross-linking buffer (1% SDS, 0.1 m NaHCO3) during 6 h at 65°C. DNA was recovered with a QIAquick PCR purification kit (Qiagen) following the manufacturer’s recommendations and quantified by quantitative real-time PCR. The primers shown in online supplemental Table 4 were used to amplify genomic regions of interest.

Statistical Analysis

GraphPad Prism 6.0 software was used to perform all statistical analyses. The data were analyzed using either one- or two-way ANOVA, or two-tailed Student’s t-test for data with normal distribution, or the nonparametric Kruskal Wallis-Dunn’s test or Mann-Whitney U test for data not following normal distribution. To make different experiments comparable, the data were normalized using the following procedure. The intensity of each experiment (ie) was calculated by determining the mean value of gene expression between the negative and positive controls. The intensities of separate experiments were normalized by the mean intensity value of all the experiments (im) and, for each experiment, the resulting normalization factor (im/ie) was multiplied by the expression levels of all the samples in that experiment.

Inflammatory Signals Modulate the Macrophage Response to Pharmacological LXR Activation

Previous work demonstrated that TLR-3 and -4 ligands, as well as IFNγ, compromised the capacity of pharmacological LXR agonists to induce key regulators of lipid metabolism [28, 29]. To gain further insight into the cross talk between inflammatory signals and the LXR pathway, we performed time-course analysis of the expression of LXR and RXR subtypes and of a battery of their well-established transcriptional targets in BMDM stimulated with the high-affinity LXR agonist T1317 and/or the inflammatory mediators TNFα, IFNγ, or LPS (Fig. 1a–c and online suppl. Fig. 1). Inflammatory signaling resulted in potent and sustained induction of Lxra expression, particularly in response to TNFα or LPS, whereas Lxrb expression was transiently upregulated by IFNγ and LPS. The effects on Lxra are in agreement with recent work demonstrating increased expression of this transcription factor upon prolonged exposure to TLR ligands [51]. In contrast with the effects on LXR subtypes, inflammatory mediators downregulated the expression of the heterodimeric partner Rxra without significantly affecting the levels of Rxrb (Fig. 1a–c). As expected, the LXR agonist did not impact the expression of LXR/RXR subtypes, but instead induced the expression of their transcriptional targets, such as Abca1, Abcg1, Srebp1c, Cd5l, and others, in a time-dependent manner (Figure 1; online suppl. Fig. 1). Notably, despite potently increasing Lxra expression, inflammatory mediators impacted negatively the induction of some LXR targets. The most drastic effects were observed on Cd5l, ApoC1, and Mertk, although the induction of Abca1 was also compromised in the presence of IFNγ (Fig. 1). Other genes, namely Abcg1 and Srebp1c, were not significantly affected by inflammatory mediators (online suppl. Fig. 1), which suggests that negative functional cross talk between inflammatory signals and the LXR pathway is gene-dependent.

Fig. 1.

Inflammatory signals modulate LXR transcriptional responses. BMDMs were treated with the LXR agonist T1317 (1 µm) (a–c), TNFα (20 ng/mL) (a), IFNγ (5 ng/mL) (b), or LPS (100 ng/mL) (c) or with the combination of both agonist and inflammatory signal for the indicated periods of time. Cells not incubated with the LXR agonist were treated with vehicle (DMSO). The expression of LXR and RXR subtypes and of LXR-RXR target genes was determined by quantitative real time PCR and normalized by the expression levels of L14. Mean ± SD of n = 4 biological replicates obtained through 2 independent experiments. Two-way ANOVA Tukey: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 vs. unstimulated cells (time 0). Significant effects of inflammatory signals on the macrophage response to the LXR agonist are also indicated: ##p < 0.01; ###p < 0.001; and ####p < 0.0001. For each of the genes analyzed, the time-course data for the LXR agonist is identical in a–c and is used as reference.

Fig. 1.

Inflammatory signals modulate LXR transcriptional responses. BMDMs were treated with the LXR agonist T1317 (1 µm) (a–c), TNFα (20 ng/mL) (a), IFNγ (5 ng/mL) (b), or LPS (100 ng/mL) (c) or with the combination of both agonist and inflammatory signal for the indicated periods of time. Cells not incubated with the LXR agonist were treated with vehicle (DMSO). The expression of LXR and RXR subtypes and of LXR-RXR target genes was determined by quantitative real time PCR and normalized by the expression levels of L14. Mean ± SD of n = 4 biological replicates obtained through 2 independent experiments. Two-way ANOVA Tukey: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 vs. unstimulated cells (time 0). Significant effects of inflammatory signals on the macrophage response to the LXR agonist are also indicated: ##p < 0.01; ###p < 0.001; and ####p < 0.0001. For each of the genes analyzed, the time-course data for the LXR agonist is identical in a–c and is used as reference.

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Notably, and in sharp contrast with the negative effects described above, inflammatory signals cooperated positively with the LXR pathway on the induction of one particular target, the gene encoding CD38 (Fig. 2a–c). This positive cross talk increased upon prolonged stimulation (Fig. 2d) and translated into strong expression of the CD38 protein (Fig. 2e), in line with our previous work [17].

Fig. 2.

Inflammatory signals and activation of the LXR/RXR pathway cooperatively induce CD38 mRNA and protein expression. BMDMs were treated with the LXR agonist T1317 (1 µm) (a–c), TNFα (20 ng/mL) (a), IFNγ (5 ng/mL) (b), or LPS (100 ng/mL) (c) or with the combination of both agonist and inflammatory signal for the indicated periods of time. d BMDMs were treated for 24 h with the same stimuli as in a and c. In a–d, cells not incubated with the LXR agonist were treated with vehicle (DMSO). The expression of Cd38 was determined by quantitative real time PCR and normalized by the expression levels of L14. The time-course data for the LXR agonist are identical in a–c. a–c Mean ± SD; n = 4 biological replicates obtained through 2 independent experiments. dn = 3 independent experiments. Two-way ANOVA Tukey: *p < 0.05; **p < 0.01; and ****p < 0.0001 vs. unstimulated cells (time 0). Additional relevant comparisons are also indicated, #p < 0.05; ##p < 0.01; and ####p < 0.0001. e BMDMs were treated for 24 h with agonists for LXR and RXR (T1317 and LG268, 1 µm each, respectively), TNFα (20 ng/mL), IFNγ (5 ng/mL), or LPS (10 ng/mL) or with simultaneous combinations of LXR/RXR agonists and inflammatory signals. Control cells were treated with vehicle (DMSO). The expression of CD38 was analyzed by Western blotting. As a control of protein loading, the expression of α-tubulin was also monitored.

Fig. 2.

Inflammatory signals and activation of the LXR/RXR pathway cooperatively induce CD38 mRNA and protein expression. BMDMs were treated with the LXR agonist T1317 (1 µm) (a–c), TNFα (20 ng/mL) (a), IFNγ (5 ng/mL) (b), or LPS (100 ng/mL) (c) or with the combination of both agonist and inflammatory signal for the indicated periods of time. d BMDMs were treated for 24 h with the same stimuli as in a and c. In a–d, cells not incubated with the LXR agonist were treated with vehicle (DMSO). The expression of Cd38 was determined by quantitative real time PCR and normalized by the expression levels of L14. The time-course data for the LXR agonist are identical in a–c. a–c Mean ± SD; n = 4 biological replicates obtained through 2 independent experiments. dn = 3 independent experiments. Two-way ANOVA Tukey: *p < 0.05; **p < 0.01; and ****p < 0.0001 vs. unstimulated cells (time 0). Additional relevant comparisons are also indicated, #p < 0.05; ##p < 0.01; and ####p < 0.0001. e BMDMs were treated for 24 h with agonists for LXR and RXR (T1317 and LG268, 1 µm each, respectively), TNFα (20 ng/mL), IFNγ (5 ng/mL), or LPS (10 ng/mL) or with simultaneous combinations of LXR/RXR agonists and inflammatory signals. Control cells were treated with vehicle (DMSO). The expression of CD38 was analyzed by Western blotting. As a control of protein loading, the expression of α-tubulin was also monitored.

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C/EBPβ Is a Key Regulator of Macrophage CD38 Expression

Considering the evidences supporting the involvement of C/EBPβ in the proinflammatory response in macrophages [37, 38], we next explored the cross talk between C/EBPβ and the LXR pathway. First, we evaluated whether pharmacological activation of LXRs impacts C/EBPβ expression in BMDMs. The levels of Cebpb mRNA were measured at different time points following macrophage stimulation with TNFα, IFNγ, or LPS, either in the presence or in the absence of the LXR agonist T1317. As a control, the expression pattern of Cebpa was also determined. As shown in Figures 3a–c, inflammatory stimuli resulted in opposite effects on Cebpa and Cebpb expression: whereas inflammatory mediators induced a transient increase in Cebpb expression, the levels of Cebpa were sharply reduced under such conditions. Notably, LXR activation did not influence the expression pattern of Cebpb (or Cebpa) in response to inflammatory signaling (Fig. 3a–c). The lack of effect of the LXR pathway was further supported by the fact that inflammatory signals induced similar levels of Cebpb expression in wild-type (WT) and LXR-deficient macrophages (Fig. 3d–f).

Fig. 3.

Inflammatory signals induce Cebpb expression in an LXR-independent manner. BMDMs were treated with the LXR agonist T1317 (1 μm) (a), TNFα (20 ng/mL) (a), IFNγ (5 ng/mL) (b), or LPS (100 ng/mL) (c) or with simultaneous combinations of the LXR agonist and the inflammatory mediator (a–c) for the indicated periods of time. Control cells were treated with vehicle (DMSO). Cebpb and Cebpa mRNA levels were determined by quantitative real time PCR and normalized by the expression levels of L14. Mean ± SD of n = 4 biological replicates obtained through 2 independent experiments. Two-way ANOVA Tukey: ****p < 0.0001 vs. unstimulated cells (time 0). ns, nonsignificant differences between time-courses. The results from the LXR agonist time-course are only displayed in a. WT or LXRα/β-deficient (LXRα/β−/−) BMDMs were treated with TNFα (20 ng/mL, 3 h) (d), IFNγ (5 ng/mL, 6 h) (e), or LPS (100 ng/mL, 6 h) (f). Gene expression was measured by quantitative real time PCR. Mean ± SD of n = 3 biological replicates. Two-way ANOVA Tukey: ****p < 0.0001. g, h Inflammatory signals strongly increase the expression of C/EBPβ LAP. BMDMs were treated with TNFα (20 ng/mL), IFNγ (5 ng/mL), or LPS (100 ng/mL) for 6 or 12 h. Control cells were left unstimulated. The expression of C/EBPβ isoforms and β-tubulin was determined by immunoblotting. g Representative immunoblot of n = 3 independent experiments that showed similar results. h Relative protein expression for the 2 C/EBPβ isoforms detected, LAP (34 kDa) and LIP (20 kDa). The relative protein expression of each C/EBPβ isoform was calculated using the Image Studio Lite software and normalized to the expression values of β-tubulin. The graphs display the quantification data of the immunoblot shown in g.

Fig. 3.

Inflammatory signals induce Cebpb expression in an LXR-independent manner. BMDMs were treated with the LXR agonist T1317 (1 μm) (a), TNFα (20 ng/mL) (a), IFNγ (5 ng/mL) (b), or LPS (100 ng/mL) (c) or with simultaneous combinations of the LXR agonist and the inflammatory mediator (a–c) for the indicated periods of time. Control cells were treated with vehicle (DMSO). Cebpb and Cebpa mRNA levels were determined by quantitative real time PCR and normalized by the expression levels of L14. Mean ± SD of n = 4 biological replicates obtained through 2 independent experiments. Two-way ANOVA Tukey: ****p < 0.0001 vs. unstimulated cells (time 0). ns, nonsignificant differences between time-courses. The results from the LXR agonist time-course are only displayed in a. WT or LXRα/β-deficient (LXRα/β−/−) BMDMs were treated with TNFα (20 ng/mL, 3 h) (d), IFNγ (5 ng/mL, 6 h) (e), or LPS (100 ng/mL, 6 h) (f). Gene expression was measured by quantitative real time PCR. Mean ± SD of n = 3 biological replicates. Two-way ANOVA Tukey: ****p < 0.0001. g, h Inflammatory signals strongly increase the expression of C/EBPβ LAP. BMDMs were treated with TNFα (20 ng/mL), IFNγ (5 ng/mL), or LPS (100 ng/mL) for 6 or 12 h. Control cells were left unstimulated. The expression of C/EBPβ isoforms and β-tubulin was determined by immunoblotting. g Representative immunoblot of n = 3 independent experiments that showed similar results. h Relative protein expression for the 2 C/EBPβ isoforms detected, LAP (34 kDa) and LIP (20 kDa). The relative protein expression of each C/EBPβ isoform was calculated using the Image Studio Lite software and normalized to the expression values of β-tubulin. The graphs display the quantification data of the immunoblot shown in g.

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We next performed immunoblotting to better understand how inflammatory signaling affects the expression of the C/EBPβ protein in BMDMs (Fig. 3g–h). Three isoforms have been described for C/EBPβ, which arise from alternative translation initiation sites [30]. Two of the isoforms, liver-enriched activating protein* (LAP*, full or C/EBPβ1, ∼38 kDa) and LAP (LAP or C/EBPβ2, ∼34 kDa), are transcriptional activators, whereas liver-enriched inhibitory protein (LIP or C/EBPβ3, ∼20 kDa) lacks the transactivation domain and is a dominant-negative isoform. The main isoform detected in differentiated BMDMs was LAP, and its expression increased progressively in response to TNFα, IFNγ, or LPS (Fig. 3g–h). Of note, a concomitant increase in the levels of the inhibitory LIP isoform was also observed upon inflammatory signaling (Fig. 3g–h), consistent with a role for this molecule as a negative feedback mechanism to avoid excessive C/EBPβ activity [56].

In view of the strong upregulation of the active C/EBPβ LAP isoform during the macrophage response to different inflammatory mediators, we evaluated whether the activity of this transcription factor is important for the regulatory actions that inflammatory signals exert on the LXR pathway. As a first approach, we analyzed publicly available RNA-Seq data comparing the response to LPS in control (C/EBPβfl/fl) and C/EBPβ-deficient (LysM-Cre-C/EBPβfl/fl) brain macrophages [38]. We placed our focus on the effects of LPS on the expression of components of the LXR pathway, namely LXRs and RXRs and the set of transcriptional targets already studied in Figure 1. LPS signaling upregulated the expression of LXRα and β, while repressing the expression of RXRα and, to a lesser extent, RXRβ (Fig. 4a–b), which highly resembles the scenario observed in BMDMs (Fig. 1). In addition, LPS potently downregulated the expression of Abca1 in brain macrophages (Fig. 4a, c). All these changes, however, occurred regardless of C/EBPβ expression. Strikingly, LPS induced a strong increase in the expression of CD38, which was fully dependent on functional C/EBPβ (Fig. 4a, c).

Fig. 4.

C/EBPβ is a key regulator of CD38 expression in brain macrophages. Analysis of RNA-seq data from primary microglial cell cultures from Cebpbfl/fl and LysMCre-Cebpbfl/fl mice treated with vehicle (VEH) or LPS (100 ng/mL) for 6 h. a Heatmap of normalized expression values of LXR and RXR subtypes, and of LXR-RXR target genes. As a control, the expression of Cebpb is also included in the analysis. For better visualization of the results, expression values of LXR and RXR subtypes (b) and of selected LXR-RXR targets (c) are also represented as graphs. Two-way ANOVA-Tukey post hoc test. Comparisons between the indicated conditions: *p < 0.05; **p < 0.01; and ***p < 0.001. Comparisons vs. the same treatment in Cebpbfl/fl cells: #p < 0.05; ##p < 0.01; and ###p < 0.001.

Fig. 4.

C/EBPβ is a key regulator of CD38 expression in brain macrophages. Analysis of RNA-seq data from primary microglial cell cultures from Cebpbfl/fl and LysMCre-Cebpbfl/fl mice treated with vehicle (VEH) or LPS (100 ng/mL) for 6 h. a Heatmap of normalized expression values of LXR and RXR subtypes, and of LXR-RXR target genes. As a control, the expression of Cebpb is also included in the analysis. For better visualization of the results, expression values of LXR and RXR subtypes (b) and of selected LXR-RXR targets (c) are also represented as graphs. Two-way ANOVA-Tukey post hoc test. Comparisons between the indicated conditions: *p < 0.05; **p < 0.01; and ***p < 0.001. Comparisons vs. the same treatment in Cebpbfl/fl cells: #p < 0.05; ##p < 0.01; and ###p < 0.001.

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C/EBPβ and Pharmacological LXR Activation Cooperate to Induce CD38 Expression

We next extended the analysis and evaluated the consequences of C/EBPβ deficiency in cells undergoing prolonged pharmacological activation of the LXR pathway. For this, BMDMs were obtained from C/EBPβfl/fl and LysM-Cre-C/EBPβfl/fl mice and then treated with the LXR agonist T1317 for 24 h either alone or in combination with proinflammatory mediators (Fig. 5). As in cells exposed to shorter time-courses (Fig. 1a–c), the LXR agonist did not affect the expression of LXR and RXR subtypes, but it did induce the expression of their transcriptional targets as expected, including Abca1, Abcg1, Srebp1c, and others. Consistent with the effects of LPS in brain macrophages (Fig. 4), stimulation of BMDMs with LPS or TNFα increased LXRα expression, while reducing the levels of RXRα, and these effects were independent of C/EBPβ (Fig. 5). In the same line, the inhibitory actions of inflammatory mediators on specific targets of the LXR pathway, namely ApoC1, Mertk, and Cd5l, were not reverted in the absence of C/EBPβ (Fig. 5). However, the induction of Cd38 by inflammatory signals either alone or in combination with the LXR agonist was drastically impaired in C/EBPβ-deficient cells (Fig. 5). These observations indicate that C/EBPβ is required for the cooperative actions that LXRs and inflammatory signaling selectively exert on CD38 expression. Of note, C/EBPβ deficiency resulted in increased basal expression of Cd38 in unstimulated BMDMs (Fig. 5), but not in microglia (Fig. 4), which is suggestive of basal derepression of this gene in BMDMs lacking functional C/EBPβ.

Fig. 5.

C/EBPβ mediates the cooperative effects between inflammatory signals and LXRs on Cd38 induction. Cebpbfl/fl and LysMCre-Cebpbfl/fl BMDMs were treated with vehicle (DMSO) or an LXR agonist (T1317, 1 µm) and/or an inflammatory stimulus, TNFα (20 ng/mL), IFNγ (5 ng/mL), or LPS (100 ng/mL) for 24 h. The expression of selected genes was measured by quantitative real time PCR and normalized by L14 expression. Mean ± standard deviation (SD) of n = 4 independent experiments performed with biological duplicates or triplicates. Two-way ANOVA Tukey (for datasets with normal distribution and homogeneous variance); Kruskal-Wallis-Dunn’s test (for datasets without normal distribution). Comparisons between the indicated conditions: *p < 0.05; **p < 0.01; and ***p < 0.001. Comparisons vs. the same treatment in Cebpbfl/fl cells: #p < 0.05; ##p < 0.01; and ###p < 0.001. For some genes, the effect of the LXR agonist was also compared using a Student’s t-test (if normal distribution) or a Mann-Whitney test (otherwise): ^p < 0.05; ^ ^p < 0.01; and ^ ^ ^p < 0.001.

Fig. 5.

C/EBPβ mediates the cooperative effects between inflammatory signals and LXRs on Cd38 induction. Cebpbfl/fl and LysMCre-Cebpbfl/fl BMDMs were treated with vehicle (DMSO) or an LXR agonist (T1317, 1 µm) and/or an inflammatory stimulus, TNFα (20 ng/mL), IFNγ (5 ng/mL), or LPS (100 ng/mL) for 24 h. The expression of selected genes was measured by quantitative real time PCR and normalized by L14 expression. Mean ± standard deviation (SD) of n = 4 independent experiments performed with biological duplicates or triplicates. Two-way ANOVA Tukey (for datasets with normal distribution and homogeneous variance); Kruskal-Wallis-Dunn’s test (for datasets without normal distribution). Comparisons between the indicated conditions: *p < 0.05; **p < 0.01; and ***p < 0.001. Comparisons vs. the same treatment in Cebpbfl/fl cells: #p < 0.05; ##p < 0.01; and ###p < 0.001. For some genes, the effect of the LXR agonist was also compared using a Student’s t-test (if normal distribution) or a Mann-Whitney test (otherwise): ^p < 0.05; ^ ^p < 0.01; and ^ ^ ^p < 0.001.

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Accumulated evidence suggests that genomic regions functioning as transcriptional enhancers are enriched in closely spaced binding sites for LDTFs and signal-dependent transcription factors and are primarily responsible for transcriptional responses to external stimuli [36]. By analyzing publicly available ChIP-Seq experiments for H3K27ac occupancy [48, 49], a histone modification that marks active enhancers [57], we identified potential transcriptional enhancers in the vicinity of the Cd38 gene that are activated in BMDMs treated with LPS (Fig. 6a, orange tracks), hereafter named regions R1, R2, and R3. Two of these regions (R1 and R2) are located upstream of the Cd38 gene, whereas region R3 is intronic. Interestingly, several putative binding motifs for LXR-RXR heterodimers (LXREs) and for C/EBPβ were identified in close proximity within these three regions (Supplemental Fig. 2).

Fig. 6.

C/EBPβ and LXR co-occupy several regions with enhancer activity upstream and within the Cd38 gene. a Analysis of ChIP-Seq data to test chromatin occupancy of H3K27ac (orange), LXRα (green), RXR (gray), and C/EBPβ (red) in the vicinity of the Cd38 gene. The occupancy of H3K27ac was analyzed in BMDMs treated with LPS for the indicated times (2 h and 4 h in database GSE56123 and 24 h in database GSE38377). LXRα ChIP-Seq was carried out using an antibody against LXRα/β in LXRβ-deficient BMDMs stimulated for 24 h with the LXR agonist GW3965 (1 μm) either alone or in combination with LPS (100 ng/mL) (database GSE200922). Binding events of RXR were determined in BMDMs treated with an LXR agonist (GW3965) for 1 h (database SRP019970). The occupancy of CEBPβ was analyzed in BMDMs from C57BL/6 mice stimulated with Kdo2-Lipid A (KLA) for 1 h (database GSE109965). Control cells were left untreated in all datasets. Three regions (R1, R2, and R3) with pronounced H3K27ac marks and LXRE and C/EBPβ binding motifs are displayed. b BMDMs were treated with an LXR agonist (T1317, 1 μm), LPS (100 ng/mL), or both stimuli simultaneously during 24 h. Control cells were treated with vehicle (DMSO). The binding of LXRs, RXRβ, and C/EBPβ to enhancer regions R1, R2, and R3 was measured by ChIP assay. One-way ANOVA: *p < 0.05; **p < 0.01; and ***p < 0.001 between the indicated conditions.

Fig. 6.

C/EBPβ and LXR co-occupy several regions with enhancer activity upstream and within the Cd38 gene. a Analysis of ChIP-Seq data to test chromatin occupancy of H3K27ac (orange), LXRα (green), RXR (gray), and C/EBPβ (red) in the vicinity of the Cd38 gene. The occupancy of H3K27ac was analyzed in BMDMs treated with LPS for the indicated times (2 h and 4 h in database GSE56123 and 24 h in database GSE38377). LXRα ChIP-Seq was carried out using an antibody against LXRα/β in LXRβ-deficient BMDMs stimulated for 24 h with the LXR agonist GW3965 (1 μm) either alone or in combination with LPS (100 ng/mL) (database GSE200922). Binding events of RXR were determined in BMDMs treated with an LXR agonist (GW3965) for 1 h (database SRP019970). The occupancy of CEBPβ was analyzed in BMDMs from C57BL/6 mice stimulated with Kdo2-Lipid A (KLA) for 1 h (database GSE109965). Control cells were left untreated in all datasets. Three regions (R1, R2, and R3) with pronounced H3K27ac marks and LXRE and C/EBPβ binding motifs are displayed. b BMDMs were treated with an LXR agonist (T1317, 1 μm), LPS (100 ng/mL), or both stimuli simultaneously during 24 h. Control cells were treated with vehicle (DMSO). The binding of LXRs, RXRβ, and C/EBPβ to enhancer regions R1, R2, and R3 was measured by ChIP assay. One-way ANOVA: *p < 0.05; **p < 0.01; and ***p < 0.001 between the indicated conditions.

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We next analyzed the binding of LXRα to these enhancers by ChIP-Seq. In these experiments, BMDMs were treated for 24 h with an LXR agonist (GW3965) either alone or in combination with LPS, whereas control cells were left untreated. Binding of LXRα increased in regions R1 and R3 in response to the LXR agonist (Fig. 6a, green tracks). Interestingly, the combination of the LXR agonist and LPS further enhanced the occupancy by LXRα in all three enhancer regions (Fig. 6a, green tracks). The binding of its heterodimeric partner RXR to these regions was also evident when we analyzed publicly available ChIP-Seq data from macrophages treated with the LXR agonist for 1 h [47] (Fig. 6a, gray track). Altogether, these data suggest that LXR-RXR heterodimers are able to bind to at least three enhancer regions that show responsiveness to LPS in the vicinity of the Cd38 gene.

Next, we used available ChIP-Seq data on C/EBPβ occupancy [46] to evaluate whether C/EBPβ has the potential to bind to the enhancer regions identified in this study. The tracks analyzed here correspond to BMDMs from C57BL/6 mice either left untreated or stimulated for 1 h with Kdo2-Lipid A, an LPS substructure with endotoxin activity analogous to that of native LPS. The occupancy of R2 by C/EBPβ was rather discrete under the conditions tested in these experiments; however, considerable binding of C/EBPβ to regions R1 and R3 was observed under basal conditions and further enhanced upon stimulation with Kdo2-Lipid A (Fig. 6a, red tracks).

Independent validation studies were performed by ChIP in order to further compare, under the same conditions, the binding of the LXR-RXR heterodimer and C/EBPβ to the three regions of interest (Fig. 6b). In these studies, WT BMDMs were treated with the LXR agonist T1317 and/or LPS for 24 h. To monitor LXR binding, we used a polyclonal antibody recognizing both LXRα and β, previously validated for ChIP assays [29, 51], whereas RXR recruitment was determined with an antibody against the RXRβ subtype. The binding of C/EBPβ was evaluated with specific antibodies against this isoform. Interestingly, C/EBPβ recruitment was observed in the three regions R1, R2, and R3 under the effect of LPS (Fig. 6b), in line with the augmented expression of this transcription factor in LPS-treated macrophages (Fig. 3). Notably, the levels of occupancy by C/EBPβ were very high in region R3, in line with the data from ChIP-Seq (Fig. 6a), which can be explained by the presence of several C/EBPβ binding sites in this region (online suppl. Fig. 2). The combination of LXR agonist and LPS did not further increase C/EBPβ recruitment to any of the regions evaluated. In contrast, the combination of signals did result in a significant enrichment of LXRs and RXRβ on the three enhancer regions as compared to their binding in basal conditions (Fig. 6b), thus validating the results observed for LXRα in ChIP-Seq (Fig. 6a). Of note, LPS treatment alone also promoted LXR/RXR binding to the R2 region. Taken together, these results suggest that combined treatment with the LXR agonist and LPS helps stabilize the LXR/RXR heterodimer in enhancer regions co-occupied by C/EBPβ upstream or within the Cd38 gene.

To further evaluate the functional cooperation between C/EBPβ and the LXR/RXR heterodimer, we performed gene reporter studies overexpressing both pathways in the absence of inflammatory signals. In these studies, we focused on region R2 for several reasons. First, we had already identified a functional LXRE within this region that was important for the cooperation between LPS signaling and the LXR pathway [17]. Second, region R2 contains only one putative C/EBPβ binding site, as opposed to the presence of several sites in regions R1 and R3, simplifying the process of introducing inactivating mutations. In a first approach, the consequences of overexpressing C/EBPβ and/or LXR/RXR were evaluated in Raw264.7 macrophages. The cells were co-transfected with the luciferase reporter plasmid pGL3-R2 along with plasmids overexpressing C/EBPβ, LXRα, LXRβ, or RXRα. Control cells were transfected with an empty plasmid (not encoding these transcription factors). Overexpression of LXR/RXR heterodimers alone resulted in a moderate increase in reporter activity, but a pharmacological agonist (GW3965) was not sufficient to upregulate this response (Fig. 7a). On the other hand, overexpression of C/EBPβ alone substantially increased reporter activity; however, this activity was further enhanced in cells co-expressing LXR/RXR heterodimers (Fig. 7a), indicating that both pathways functionally cooperate with each other to promote reporter activity. Interestingly, pharmacological activation of LXRs further increased the reporter activity only in cells co-overexpressing C/EBPβ and LXR/RXR. For this reason, subsequent studies were carried out with the overexpression of these molecules.

Fig. 7.

C/EBPβ and LXRs functionally cooperate in a genomic region with enhancer activity. a–f Luciferase reporter studies using a pGL3 luciferase reporter vector containing the enhancer region R2 (pGL3-R2). Raw264.7 macrophages (a–e) or COS-7 kidney cells (f) were transiently co-transfected with pGL3-R2 containing either a WT sequence (a–f) or a mutated (MUT) LXRE (b, e, f) or C/EBPβ binding site (c–e). In addition, some transfections included pMSV-C/EBPβ (for C/EBPβ overexpression) (a–f), and/or a combination of pcDNA3-RXRα and either pcDNA3-LXRα (a, e) or pcDNA3-LXRβ (a, b, d, f) (for LXR/RXR overexpression). In a, control cells were transfected with pGL3-R2 and an empty plasmid (empty pcDNA3 without overexpression of transcription factors). All transfections included pRL-TK (for constitutive Renilla expression) and the total amount of plasmid DNA was equilibrated using empty pcDNA3. The cells were then stimulated with an LXR agonist (GW3965, 1 µm) or vehicle for 24 h. The enhancer activity is represented as luciferase activity normalized by Renilla activity. Mean ± SD of biological triplicates (a, c, f) or of n = 3 independent experiments each performed with biological duplicates or triplicates (b, d–e). a–f Two-way ANOVA: *p < 0.05; **p < 0.01; and ***p < 0.001 between the indicated conditions; in addition, #p < 0.05; ##p < 0.01; and ###p < 0.001 in comparison with the same condition in control cells not overexpressing transcription factors (empty plasmid) (a) or in comparison with the same condition in cells transfected with the WT pGL3-R2 sequence (b, d–f).

Fig. 7.

C/EBPβ and LXRs functionally cooperate in a genomic region with enhancer activity. a–f Luciferase reporter studies using a pGL3 luciferase reporter vector containing the enhancer region R2 (pGL3-R2). Raw264.7 macrophages (a–e) or COS-7 kidney cells (f) were transiently co-transfected with pGL3-R2 containing either a WT sequence (a–f) or a mutated (MUT) LXRE (b, e, f) or C/EBPβ binding site (c–e). In addition, some transfections included pMSV-C/EBPβ (for C/EBPβ overexpression) (a–f), and/or a combination of pcDNA3-RXRα and either pcDNA3-LXRα (a, e) or pcDNA3-LXRβ (a, b, d, f) (for LXR/RXR overexpression). In a, control cells were transfected with pGL3-R2 and an empty plasmid (empty pcDNA3 without overexpression of transcription factors). All transfections included pRL-TK (for constitutive Renilla expression) and the total amount of plasmid DNA was equilibrated using empty pcDNA3. The cells were then stimulated with an LXR agonist (GW3965, 1 µm) or vehicle for 24 h. The enhancer activity is represented as luciferase activity normalized by Renilla activity. Mean ± SD of biological triplicates (a, c, f) or of n = 3 independent experiments each performed with biological duplicates or triplicates (b, d–e). a–f Two-way ANOVA: *p < 0.05; **p < 0.01; and ***p < 0.001 between the indicated conditions; in addition, #p < 0.05; ##p < 0.01; and ###p < 0.001 in comparison with the same condition in control cells not overexpressing transcription factors (empty plasmid) (a) or in comparison with the same condition in cells transfected with the WT pGL3-R2 sequence (b, d–f).

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We next evaluated the consequences of mutating specific response elements. Mutations in a bona fide LXRE blunted both the response to C/EBPβ and the cooperative effect between C/EBPβ and the LXR pathway (Fig. 7b). Likewise, a mutated version of the C/EBPβ binding site impaired the response to C/EBPβ when acting alone (Fig. 7c) or in combination with activated LXR/RXR (Fig. 7d), indicating that synergistic cooperation between C/EBPβ and LXR/RXR requires binding sites for both pathways. Similar results were obtained when LXRβ (Fig. 7b, d) or LXRα (Fig. 7e) was used in these studies. In contrast with the results obtained in Raw264.7 cells, C/EBPβ overexpression was not sufficient to promote reporter activity in COS-7 kidney cells (Fig. 7f), suggesting that lineage-dependent factors are required for the activity of C/EBPβ on the R2 enhancer.

Taken together, these results strongly suggest that C/EBPβ and LXR/RXR bind to several regions with enhancer activity upstream or within the Cd38 gene. In addition, gene-reporter assays further support the existence of functional synergistic cooperation between these two pathways in macrophages to control Cd38 transcription.

CD38 Activity Is Involved in the Fine-Tuning of Cytokine Production in Macrophages

Based on the essential role of C/EBPβ in the regulation of CD38 expression in macrophages, we investigated whether these two molecules are coordinately involved in the macrophage response to LPS. To answer this question, we reanalyzed the RNA-Seq data from Cebpbfl/fl and LysM-Cre-Cebpbfl/fl brain macrophages [38] in search of genes induced by LPS that are highly dependent on C/EBPβ. To this end, validated protein-coding genes were filtered based on two conditions: (1) expression induced by LPS at least 3-fold (log2 fold change > 1.58, with an adjusted p value < 0.001) in control Cebpbfl/fl macrophages, and (2) expression drastically reduced in LPS-treated LysMCre-Cebpbfl/fl macrophages compared to LPS-treated control Cebpbfl/fl macrophages (log2 fold change < −3, with an adjusted p value < 0.001). A list of C/EBPβ-dependent genes is displayed in Figure 8a, ordered by their log2 fold change between LPS-treated C/EBPβ-deficient macrophages and LPS-treated control cells. In addition, genes encoding for cytokines and other classical mediators of the macrophage response to LPS were included in the analysis (Fig. 8b). One of these genes codes for cholesterol 25-hydroxylase (Ch25h), which catalyzes the production of 25-hydroxycholesterol (25-HC), a natural endogenous LXR ligand. With the exception of Il12b, the genes analyzed here showed a tendency for downregulation in C/EBPβ-dependent cells (Fig. 8b).

Fig. 8.

CD38 is dispensable for the induction of C/EBPβ-dependent genes, but contributes to fine-tuning macrophage cytokine production in response to LPS. a RNA-Seq data from Cebpbfl/fl and LysMCre-Cebpbfl/fl brain macrophages treated with vehicle or LPS (100 ng/mL) for 6 h were analyzed to identify LPS-induced genes that are highly dependent on C/EBPβ expression. Heatmap displaying a list of filtered genes ordered by the log2 fold change between LPS-treated LysMCre-Cebpbfl/fl vs. LPS-treated Cebpbfl/fl macrophages. b Graph representing log2 fold changes in the expression of selected cytokines and inflammatory mediators in LPS-treated LysMCre-Cebpbfl/fl vs. LPS-treated Cebpbfl/fl macrophages. c-e BMDMs obtained from WT or CD38-deficient mice were treated with LPS (100 ng/mL) during the indicated periods of time. In c, the expression of selected genes, including C/EBPβ-dependent genes identified in a, was analyzed by quantitative real time PCR. The levels of secreted IL-6 (d) and IL-12 (e) were measured by ELISA. Mean ± SD; n = 3 experiments. Two-way ANOVA Tukey: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 vs. unstimulated cells (time 0). Significant changes between WT- and CD38-deficient cells are also indicated: #p < 0.05; ##p < 0.01; and ###p < 0.001.

Fig. 8.

CD38 is dispensable for the induction of C/EBPβ-dependent genes, but contributes to fine-tuning macrophage cytokine production in response to LPS. a RNA-Seq data from Cebpbfl/fl and LysMCre-Cebpbfl/fl brain macrophages treated with vehicle or LPS (100 ng/mL) for 6 h were analyzed to identify LPS-induced genes that are highly dependent on C/EBPβ expression. Heatmap displaying a list of filtered genes ordered by the log2 fold change between LPS-treated LysMCre-Cebpbfl/fl vs. LPS-treated Cebpbfl/fl macrophages. b Graph representing log2 fold changes in the expression of selected cytokines and inflammatory mediators in LPS-treated LysMCre-Cebpbfl/fl vs. LPS-treated Cebpbfl/fl macrophages. c-e BMDMs obtained from WT or CD38-deficient mice were treated with LPS (100 ng/mL) during the indicated periods of time. In c, the expression of selected genes, including C/EBPβ-dependent genes identified in a, was analyzed by quantitative real time PCR. The levels of secreted IL-6 (d) and IL-12 (e) were measured by ELISA. Mean ± SD; n = 3 experiments. Two-way ANOVA Tukey: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 vs. unstimulated cells (time 0). Significant changes between WT- and CD38-deficient cells are also indicated: #p < 0.05; ##p < 0.01; and ###p < 0.001.

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We then determined whether C/EBPβ-dependent genes also showed dependency on CD38 expression. For this, BMDMs were obtained from WT and CD38-deficient mice and treated with LPS for different periods of time (Fig. 8c). The expression of Il6 and Il12b was significantly reduced in CD38-deficient macrophages (Fig. 8c), which translated in reduced secretion of these cytokines (Fig. 8d–e). These observations are in line with previous data from our group, showing decreased cytokine expression in CD38-deficient macrophages undergoing bacterial infection [17]. However, while these results support a proinflammatory role for CD38 in contributing to fine-tuning the production of certain cytokines by macrophages, the majority of the C/EBPβ-dependent genes analyzed here were not affected by the absence of functional CD38 (Fig. 8c), suggesting that CD38 is not required for the overall C/EBPβ transcriptional response.

CD38 displays receptor and enzymatic activities that facilitate the establishment of an effective immune response during infection [20]. However, recent studies also suggest the involvement of the enzymatic activity of CD38 in immunosuppression, for example, within the tumor microenvironment [58]. Several of its activities use large amounts of NAD as a substrate, which translates into CD38 being the main NAD-degrading enzyme in mammalian tissues [21]. Therefore, the modulation of the levels or the activity of CD38 offers therapeutic potential, especially in diseases associated with NAD decline [59].

The study presented here contributes to the understanding of the mechanisms governing the expression of CD38 by uncovering the essential role of C/EBPβ in the transcriptional regulation of the Cd38 gene. In this sense, the induction of CD38 by inflammatory mediators (TNFα, IFNγ, and LPS), either alone or in combination with pharmacological LXR activation, was severely impaired in C/EBPβ-deficient macrophages. Members of the C/EBP family bind to common elements on DNA and, in some cases, their functions overlap, as it is the case of C/EBPβ and C/EBPδ on a number of LPS-induced genes [60]. However, our results indicate that, in macrophages, C/EBPβ is indispensable for the induction of CD38 by inflammatory mediators and this role is not compensated by other members of the family. In addition, overexpression of C/EBPβ in Raw264.7 macrophages (in the absence of inflammatory signals) is sufficient to induce transcriptional activity from an enhancer region containing a C/EBPβ binding site located upstream of the Cd38 gene.

Despite reciprocal negative interactions between LXRs and inflammatory signals in different settings [26], the induction of CD38 expression stands out as being cooperatively targeted by both pathways. In this regard, another important conclusion from this study is the functional cooperation between C/EBPβ and the LXR pathway in mediating CD38 transcription, which is supported by the finding of several regions with enhancer activity near the Cd38 gene to which both transcription factors are able to bind. C/EBP family members are known to establish pioneering functions in determining enhancer selection in macrophages [35]. A hierarchical model has been proposed in which LDTFs, including PU.1 and C/EBPα/β in macrophages, collaborate in enhancer selection and priming by cooperatively interacting at genomic regions containing closely spaced binding motifs for each factor. In response to stimuli, signal-dependent transcription factors regulate gene expression in a cell type-specific manner by binding to primed enhancers [36]. Based on the pioneering roles described for C/EBPβ in macrophages [35], we anticipated that C/EBPβ deficiency would impact broadly the macrophage response to pharmacological activators of the LXR pathway. To our surprise, however, except for the case of CD38, the rest of LXR target genes evaluated here (including canonical targets such as Abca1, Abcg1, and Srebp1c) were induced by an LXR agonist at similar levels in control and C/EBPβ-deficient macrophages. We cannot discard here that C/EBPα might be able to compensate for the lack of C/EBPβ and facilitate LXR-mediated transcription of these genes, especially in the absence of inflammatory signals (when C/EBPα is highly expressed). In this sense, differentiation of specific populations of resident peritoneal and lung macrophages is highly sensitive to the absence of functional C/EBPβ, but other tissue macrophages develop normally in C/EBPβ-deficient mice [61], indicating that C/EBPβ is dispensable at the steady state in many macrophage populations.

C/EBPα and C/EBPβ were indeed oppositely regulated in response to inflammatory mediators, which is in line with previous observations [62], but neither activation nor deletion of LXRs impacted their expression profile. Likewise, basal expression of LXR or RXR isoforms in macrophages was not substantially affected by C/EBPβ deficiency, indicating that cooperation between these transcription factors to activate CD38 transcription does not require reciprocal regulation of their expression levels. The results shown here may also have physiological implications when LXR agonists are produced endogenously. Several evidences demonstrate accumulation of the natural LXR agonist 25-HC (as a consequence of the increase in CH25H expression) during the inflammatory response (reviewed in [63]). In fact, in our previous work, we showed synergistic effects between inflammatory signaling and 25-HC on Cd38 expression in an LXR-dependent manner [17]. In the gene reporter studies presented in this work, the mere overexpression of LXR/RXR heterodimers (in the absence of pharmacological LXR activation) resulted in moderate induction of the enhancer activity of region R2 (Fig. 7a). This activity was further enhanced upon addition of C/EBPβ to the system, which suggests that endogenous agonists of the LXR/RXR pathway might be produced during transfection of Raw264.7 cells.

The strong increase in C/EBPβ expression in response to inflammatory mediators, in particular the active LAP isoform, has been shown to be important for the upregulation of specific subsets of inflammatory genes in activated macrophages [30, 64]. In line with this notion, mice with C/EBPβ-deficient myeloid cells showed robust attenuation of the clinical signs associated to experimental autoimmune encephalitis [38]. In this work, we analyzed RNA-Seq data to identify genes strongly induced by LPS in a C/EBPβ-dependent manner. Some of these genes were also shown to be C/EBPβ-dependent in a previous study [64]. Comparative analysis of LPS-induced gene expression in WT and CD38-deficient macrophages indicated that CD38 activity contributes to the inflammatory response by fine-tuning the production of specific cytokines, namely IL-6 and IL-12. IL-6, but not IL-12, is also a target of C/EBPβ ([64] and Fig. 8b). Our data do not support a major role for CD38 in the overall C/EBPβ transcriptional response, but impaired expression of CD38 in mice with C/EBPβ-deficient microglia may influence cytokine production and contribute to the ameliorated inflammatory phenotype of these mice.

In conclusion, the study presented here identifies C/EBPβ as an essential mechanism by which inflammatory signals and the LXR pathway synergistically upregulate the expression of the multifunctional protein CD38 in macrophages. These observations raise potential novel targets that may provide therapeutic opportunities, either alternatively or in combination with anti-CD38 antibodies, for diseases in which CD38 has pathological implications.

We thank Dr. D. Mangelsdorf for the LXR-deficient mice, Dr. F. Lund for making the CD38-deficient mice available, Dr. J. Sancho for facilitating their transfer to our group, Dr. K.R. Steffensen for antibodies against LXRs, Dr. E. Ballestar for antibodies against RXRβ, and M. García and V. González for technical assistance. Part of the materials included in this manuscript were first described by the PhD thesis defended by E.G. at the University of Barcelona.

The study was approved by the Bioethics Commission of the University of Barcelona (Institutional Review Board IRB00003099). All the protocols requiring animal manipulation were approved by the Institutional Animal Care and Use Committees from Parc Científic de Barcelona (approved by the Government of Catalonia [Generalitat de Catalunya] with number 9672) and University of Barcelona (number 222/19), following the standard ethical regulations and meeting quality and experimental requirements of current applicable National (RD 53/2013, article 38) and European legislation (RD 53/2013 Council Directive; 2010/63/UE; Order 214/1997/GC).

E.G. is currently an employee at OneChain Immunotherapeutics. J.M. is currently an employee at Avidity Biosciences. E.N.C. holds a patent on the use of CD38 inhibitors for metabolic diseases that is licensed by Elysium Health. E.N.C. is a consultant for TeneoBio, Calico, Mitobridge, and Cytokinetics. E.N.C. is on the advisory board of Eolo Pharma. E.N.C. owns stocks in TeneoBio. Dr. Chini is the head of the external research advisory board for Neolaia Bio. Research in the Chini laboratory has been conducted in compliance with the Mayo Clinic conflict of interest policies. C.C. is a consultant for Aromics. The rest of the authors declare no potential conflicts of interest.

This work was supported by Grants SAF2017-89510-R and PID2020-119875RB-I00 to A.F.V. and C.C., PID2022-137696OB-I00 to A.C., and SAF2017-90604-REDT and SAF2015-71878-REDT to the NuRCaMeIn network, funded by MICIU/AEI/10.13039/501100011033 and by ERDF A way of making Europe; grants 201605.30 to A.F.V. and 201605.31 to C.C. funded by Fundació La Marató de TV3; grants PI14/302 to J.S., and PI19/0593 and PI23/0850 to J.M.V.T., funded by Instituto de Salud Carlos III and by ERDF/EU A way to make Europe; grant from Fundación Luzón (Programa de Ayudas para Proyectos de Investigación en ELA Unzué-Luzón 2024) to J.M.V.T.; grants 2021SGR01433 and 2021SGR01121 from AGAUR-Generalitat de Catalunya to A.F.V. and J.M.V.T., respectively; and the European Cooperation in Science and Technology (COST) Actions BM1404 Mye-EUNITER and CA20117 Mye-InfoBank. E.G. received a fellowship from the University of Barcelona (APIF). J.F.D. and J.M. received fellowships PRE2018-085579 and BES-2009-014828, respectively, funded by MICIU/AEI/10.13039/501100011033 and by FSE invests in your future.

Conception and design, and writing – original draft: E.G. and A.F.V.; development of methodology: E.G., P.R.M., J.F.D., J.V.D., A.C., D.J.C., J.M., and A.F.V.; acquisition of data: E.G., J.F.D., P.R.M., J.V.D., A.C., and A.F.V.; analysis and interpretation of data: E.G., J.F.D., P.R.M., J.V.D., A.C., J.M.V.T., J.S., C.C., and A.F.V.; material support: A.C., J.S., E.N.C., and C.C.; and study supervision: A.F.V.

All data generated or analyzed during this study are included in this article. As part of the study, the authors have analyzed publicly available RNA-Seq and ChIP-Seq datasets. Further inquiries can be directed to the corresponding authors.

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