Serotonin (5-HT)

Serotonin (5-HT) Shapes the Macrophage Gene Profile through the 5-HT 2B− Dependent Activation of the Aryl Hydrocarbon Receptor This information is current as of September 29, 2021. Concha Nieto, Ignacio Rayo, Mateo de las Casas-Engel, Elena Izquierdo, Bárbara Alonso, Catherine Béchade, Luc Maroteaux, Miguel A. Vega and Ángel L. Corbí J Immunol published online 6 April 2020 http://www.jimmunol.org/content/early/2020/04/04/jimmun Downloaded from ol.1901531

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 6, 2020, doi:10.4049/jimmunol.1901531 The Journal of Immunology

Serotonin (5-HT) Shapes the Macrophage Gene Proﬁle through the 5-HT2B–Dependent Activation of the Aryl Hydrocarbon Receptor

Concha Nieto,*,1 Ignacio Rayo,*,1 Mateo de las Casas-Engel,* Elena Izquierdo,* Ba´rbara Alonso,* Catherine Be´chade,†,‡,x Luc Maroteaux,†,‡,x Miguel A. Vega,* and A´ ngel L. Corbı´*

Macrophages can either promote or resolve inﬂammatory responses, and their polarization state is modulated by peripheral serotonin (5-hydroxytryptamine [5-HT]). In fact, pro- and anti-inﬂammatory macrophages differ in the expression of serotonin

receptors, with 5-HT2B and 5-HT7 expression restricted to M-CSF–primed monocyte-derived macrophages (M-MØ). 5-HT7 drives Downloaded from the acquisition of proﬁbrotic and anti-inﬂammatory functions in M-MØ, whereas 5-HT2B prevents the degeneration of spinal cord mononuclear phagocytes and modulates motility of murine microglial processes. Because 5-HT2B mediates clinically relevant 5-HT–related pathologies (valvular heart disease, pulmonary arterial hypertension) and is an off target of anesthetics, antipar-

kinsonian drugs, and selective serotonin reuptake inhibitors, we sought to determine the transcriptional consequences of 5-HT2B engagement in human macrophages, for which 5-HT2B signaling remains unknown. Assessment of the effects of specific agonists and antagonist revealed that 5-HT2B engagement modifies the cytokine and gene signature of anti-inflammatory M-MØ, upregulates the expression of aryl hydrocarbon receptor (AhR) target genes, and stimulates the transcriptional activation of AhR. http://www.jimmunol.org/ Moreover, we found that 5-HT dose dependently upregulates the expression of AhR target genes in M-MØ and that the 5-HT–

mediated activation of AhR is 5-HT2B dependent because it is abrogated by the 5-HT2B–speciﬁc antagonist SB204741. Altogether, our results demonstrate the existence of a functional 5-HT/5-HT2B/AhR axis in human macrophages and indicate that 5-HT potentiates the activity of a transcription factor (AhR) that regulates immune responses and the biological responses to xenobiotics. The Journal of Immunology, 2020, 204: 000–000.

eripheral serotonin (5-hydroxytryptamine [5-HT]) inﬂu- Macrophages promote the initiation and the resolution of in-

ences the physiopathology of numerous tissues (1–3), as it flammatory processes, with both processes being essential for by guest on September 29, 2021 P controls vascular, heart, and gastrointestinal functions (4, 5), maintaining tissue homeostasis (20, 21). In fact, modulation of promotes cell proliferation (6, 7), regulates wound healing, and in- macrophage polarization is an attractive therapeutic target for fluences immune and inflammatory responses by modulating chronic inflammatory pathologies, in which the balance between T lymphocyte and myeloid cell functions (8, 9). In inflammatory proinflammatory and resolving macrophages is altered (22). Not pathologies, 5-HT contributes to pulmonary arterial hypertension surprisingly, macrophage effector functions are directly modulated (10), atopic dermatitis (11), systemic sclerosis (12), inflammatory by 5-HT (8–10, 16) and also by selective serotonin reuptake in- gut disorders (13–15), cancer angiogenesis (16), and neuroendocrine hibitors (SSRI) (23, 24). We have previously demonstrated that neoplasms proliferation (17), whereas it reduces pathologic scores 5-HT7 is the major mediator of the anti-inflammatory actions of in collagen-induced arthritis (18). The existence of seven types of 5-HT on human macrophages (25) and that both 5-HT2B and 5-HT7 5-HT receptors (5-HT1–7) with different distribution and signaling contribute to the maintenance of the anti-inflammatory state of properties (19) underlies these tissue-specific actions of 5-HT. M-CSF–dependent human macrophages (26).

*Laboratorio de Ce´lulas Mieloides, Centro de Investigaciones Biolo´gicas, Consejo The microarray data presented in this article have been submitted to the Gene Superior de Investigaciones Cientı´ficas, 28040 Madrid, Spain; †INSERM UMR-S839, Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number 75005 Paris, France; ‡Sorbonne University and UPMC University Paris, 75005 Paris, GSE121825. France; and xInstitut du Fer a` Moulin, 75005 Paris, France Address correspondence and reprint requests to Dr. Concha Nieto and Dr. A´ ngel 1C.N. and I.R. contributed equally to this work, and the order of authors is arbitrary. L. Corbı´, Myeloid Cell Laboratory, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientifıćas, Calle Ramiro de Maeztu, 9, 28040 Madrid, ORCIDs: 0000-0003-0790-3440 (C.N.); 0000-0002-9499-8603 (L.M.); 0000-0001- Spain. E-mail addresses: [email protected] (C.N.) and [email protected] (A´ .L.C.) 6151-4193 (M.A.V.). The online version of this article contains supplemental material. Received for publication December 30, 2019. Accepted for publication March 16, 2020. Abbreviations used in this article: adj, adjusted; AhR, aryl hydrocarbon receptor; AMS, a-methylserotonin; DGE, differential gene expression; GO, gene ontology; GSEA, This work was supported by grants from Ministerio de Ciencia e Innovacioń gene set enrichment analysis; 5-HT, 5-hydroxytryptamine; M-MØ, M-CSF–primed (SAF2014-52423-R and SAF2017-83785-R) (to M.A.V. and A´ .L.C.), 201619.31 monocyte-derived macrophage; PKA, protein kinase A; SERT, serotonin transporter; from Fundacio´ La Marato´/TV3 (to A´ .L.C.), and Red de Investigacioń en Enferme- siAhR, AhR-specific small interfering RNA; siC, control small interfering RNA; SSRI, dades Reuma´ticas (RD16/0012/0007), and cofinanced by the European Regional selective serotonin reuptake inhibitor; XRE, xenobiotic response element. Development Fund “A way to achieve Europe.” M.d.l.C.-E. and I.R. were funded ´ by a Formacion de Personal Investigador predoctoral fellowship (BES-2009-021465 Ó and BES-2015-071337, respectively) from Ministerio de Ciencia e Innovacioń. Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 C.N., I.R., M.d.l.C.-E., E.I., B.A., and C.B. performed research and analyzed data; C.N., I.R., M.d.l.C.-E., C.B., L.M., M.A.V., and A´ .L.C. designed the research and analyzed data; A´ .L.C. wrote the paper.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901531 2 5-HT2B SHAPES THE MACROPHAGE TRANSCRIPTOME VIA AhR

The 5-HT2B serotonin receptor is expressed in the CNS, in which AhR-binding element (XRE), was used for the reporter gene assays. For it exerts antidepressant or anxiolytic-like effects (27), and in pe- normalizing transfection efficiency, transfected DNA included a 40:1 mixture riphery, in which it plays a major role in heart development (28) and of the XRE-specific firefly luciferase construct and a construct expressing Renilla luciferase from a constitutive promoter. HepG2 cells (80,000 cells per in 5-HT–mediated pathologies like pulmonary arterial hypertension well) or M-MØ (1 3 106 cells per well) were transfected with 1 mgofthe (10), dermal, lung and liver fibrosis (12, 29), cardiac hypertrophy XRE reporter construct using either Superfect (Qiagen) or VIROMER (30), and valvular heart disease associated with carcinoid syndrome RED (Lipocalix), respectively. After transfection, cells were cultured over- m (31). Importantly, antimigraine drugs like methysergide and night before exposure to BW723C86 (10 M), FICZ (250 nM), CH223191 (3 mM), or DMSO for 24 h and lysed; firefly and Renilla luciferase activities ergotamine (32), general anesthetics (33), conventional SSRIs were determined using the Dual-Luciferase Reporter Assay System (Promega). (34), and even antiparkinsonian dopamine agonists (35) exert Small interfering RNA transfection potent off-target effects on 5-HT2B (36). Because some of these 6 5-HT2B–targeting drugs induce valvular heart disease, their use is To knockdown AhR expression, M-MØ (1 3 10 cells) were transfected restricted, and novel potential drugs are commonly screened for with AhR-specific small interfering RNA (siAhR; 50 nM) (no. s1199; Thermo Fisher Scientific) using HiPerFect Transfection Reagent (Qiagen). 5-HT2B agonist activity. A negative control small interfering RNA (siC; no. 4390843; Thermo 5-HT2B engagement enhances proliferation of numerous cell a Fisher Scientific) was used as a control. Cells were allowed to recover from types via G q and Src phosphorylation and through production of transfection in complete medium (18–24 h) and then treated for 6 h with growth factors like insulin, TGFb1, CTGF, FGF2 and TGFa (37). BW723C86 (10 mM) before analysis. In macrophages, 5-HT2B prevents mononuclear phagocyte de- Real-time quantitative RT-PCR generation in amyotrophic lateral sclerosis (38), and Htr2b2/2 microglia exhibits a mild inflammatory state (39), which is in line Total RNA was extracted using the Total RNA and Protein Isolation Kit Downloaded from (Macherey-Nagel). RNA samples were retrotranscribed (High-Capacity with the ability of 5-HT2B antagonist SB204741 to impair the ac- cDNA Reverse Transcription Kit; Applied Biosystems), and triplicates of quisition of human macrophage polarization-specific genes (26) and amplified cDNA were analyzed on a Light Cycler 480. Gene-specific oli- TGFb1 expression by mouse Kupffer cells (29). However, although gonucleotides (Supplemental Table I) were designed using the Universal 5-HT2B activation modifies inflammatory cytokine production by ProbeLibrary software (Roche Diagnostics). Results were expressed rela- human monocytes (40), the functional and transcriptional conse- tive to the expression level of the endogenous reference gene TBP and using the DD cycle threshold method for quantitation. quences of 5-HT2B ligation in human macrophage remains to be http://www.jimmunol.org/ determined. Because anti-inflammatory M-CSF–dependent macro- Microarray and RNA sequencing analysis phages express 5-HT2B, we have assessed the specific role of Global gene expression analysis was performed on RNA from three in- 5-HT2B in macrophage polarization and found that both 5-HT and dependent samples of untreated (control), BW723C86-treated (10 mM), m the 5-HT2B agonist BW723C86 are capable of shaping the macro- SB204741-treated (1 M), or SB204741 + BW723C86–treated M-MØ. phage transcriptome via aryl hydrocarbon receptor (AhR) activation. RNA was isolated using the RNeasy Mini Kit (Qiagen) and used a hy- bridization probe on Whole Human Genome Microarrays (Agilent Tech- nologies, Palo Alto, CA). Only probes with signal values .60% quantile in Materials and Methods at least one condition were considered for the differential gene expression (DGE) and statistical analysis. Statistical analysis for DGE was carried out

Generation of human monocyte-derived macrophages and using empirical Bayes moderated paired t test implemented in the limma by guest on September 29, 2021 cell culture package (http://www.bioconductor.org), and p values were adjusted for Buffy coats from anonymous healthy blood donors were provided by multiple-hypotheses testing using the Benjamini-Hochberg method to control Comunidad de Madrid Blood Bank. Ethical approvals for all blood processes the false discovery rate (45), with all procedures coded in R (http://www.r- used in this study were approved by the Centro de Investigaciones Biolo´gicas project.org). Microarray data were deposited in the Gene Expression Omnibus Ethics Committee, and all experiments were carried out in accordance with (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE121825. RNA sequencing (RNAseq) was performed on three independent samples of M-MØ the approved guidelines and regulations. Human PBMCs were isolated from m m m buffy coats over a Lymphoprep (Nycomed Pharmaceuticals) gradient, and exposedfor6hto10 M BW723C86, 10 M 5-HT, or 100 M5-HTand monocytes were purified from PBMC by magnetic cell sorting using CD14 processed at BGI (https://www.bgitechsolutions.com) using the BGISEQ-500 microbeads (Miltenyi Biotech). Monocytes (.95% CD14+ cells) were cul- platform. An average of 5.41 Gb clean reads were generated per sample tured at 0.5 3 106 cells per milliliter for 7 d in RPMI 1640 supplemented and, after filtering, clean reads were mapped to the reference (University of with 10% FCS (Biowest) (completed medium) at 37˚C in a humidified at- California, Santa Cruz Genome Browser Gateway Assembly hg38) using Bowtie2 (average mapping ratio of 93.41%) (46). Gene expression levels mosphere with 5% CO2 and contained M-CSF (10 ng/ml) (ImmunoTools GmbH) to generate M-CSF–primed monocyte-derived macrophages were calculated by using the RNA-Seq by Expectation Minimization software package (47), and DGE was assessed by using the R-package (M-MØ). M-CSF was added every 2 d. Before treatments, M-MØ were . maintained in serum-free medium (Macrophage-SFM; Life Technologies) DESeq2 algorithms using the parameters fold change 2andadjusted (adj) p value ,0.05. Heatmaps and clustering were done using the for 48 h. For macrophage activation, cells were exposed to 5-HT2B agonists for 6 h and then treated with Escherichia coli 055:B5 LPS (10 ng/ml) for Genesis software (http://genome.tugraz.at/genesisclient/) (48). Differentially expressed genes were analyzed for annotated gene sets enrichment using 18 h. The 5-HT2B agonists BW723C86 (a-methyl-5-(2-thienylmethoxy)-1H- indole-3-ethanamine) (41, 42) and a-methylserotonin (AMS) (43) and the Enrichr (http://amp.pharm.mssm.edu/Enrichr/) (49), and enrichment terms adj p , 5-HT antagonist SB204741 (44) were purchased from Sigma-Aldrich. The were considered significant with a Benjamini-Hochberg– value 0.05. 2B For gene set enrichment analysis (GSEA) (50), gene sets available at the web 5-HT2B agonist 6-APB (6-(2-aminopropyl)benzofuran) was obtained from Cayman and used at the indicated concentrations. The AhR agonist FICZ site, as well as gene sets generated from publicly available transcriptional (Enzo) and antagonist CH223191 (Calbiochem) were used at 250 nM and 3 studies (https://www.ncbi.nlm.nih.gov/gds), were used. mM, respectively. When indicated, SB204741 was used 1 h before treatment Statistical analysis with agonists. Human TNF-a and CCL2 were measured using commercially available ELISA (BD Biosciences). M-MØ culture supernatants were tested For comparison of means, statistical significance was assessed in a pairwise for the presence of TNF-a and CCL2 using commercially available ELISA manner between pairs of experimental conditions of the generated data and sets (BD Biosciences) and following the protocol supplied by the manu- evaluated using the Student paired t test. In all cases, p , 0.05 was con- facturer. For Western blot, cell lysates (20 mg) were subjected to SDS- sidered as statistically significant. PAGE and transferred onto Immobilon polyvinylidene difluoride membrane (Millipore). After blocking with 5% nonfat dry milk, AhR detection was Results carried out with a goat polyclonal Ab (no. sc 8087; Santa Cruz Biotechnology). The BW723C86 agonist modulates the transcriptional Transfections and reporter gene assays signature of human macrophages partially via 5-HT2B

The Cignal Xenobiotic Response Element (XRE) Reporter Assay Kit Human anti-inﬂammatory M-MØ express 5-HT7 and 5-HT2B se- (Qiagen), in which the XRE-luciferase construct harbors tandem repeats of the rotonin receptors, and both contribute to the acquisition of the The Journal of Immunology 3

M-MØ–specific transcriptome (25, 26). Because 5-HT2B is the target absence or presence of the antagonist (Fig. 1C, 1D) indicated that of clinically relevant drugs (anesthetics, SSRIs, antimigraine drugs) the antagonist SB204741 prevents the BW723C86-mediated up- (32–34), we sought to determine the consequences of 5-HT2B en- regulation of 217 genes, and the BW723C86-mediated downreg- gagement in human macrophages. To that end we made use of ulation of 334 genes (Fig. 1C, 1D, Supplemental Table II). BW723C86, a 5-HT2B agonist with 10-fold and 100-fold selectivity Therefore, 5-HT2B mediates the enhanced expression of 60% of over human 5-HT2C and 5-HT2A, respectively (51), amply used as the genes upregulated by BW723C86 (217 genes) and the di- in vivo (34, 52–54) and ex vivo (18, 38, 55). Transcriptional minished expression of 84% of the genes downregulated by profiling of BW723C86-treated M-MØ (Fig. 1A) revealed that the BW723C86 (Fig. 1E, Supplemental Table II). These results also in- agonist increased the expression of 357 annotated genes and dicate the existence of 5-HT2B–independent effects of BW723C86, as downregulated the expression of 401 genes (adj p , 0.002) the agonist modulates the expression of 207 genes (140 upregulated, (Fig. 1B, Supplemental Table II). GSEA revealed that BW723C86 67 downregulated) even in thepresenceofthe5-HT2B antagonist significantly modifies the expression of Ga12/13 signaling and (Fig. 1C, 1D, Supplemental Table II). Altogether, these results amyotrophic lateral sclerosis gene sets, in agreement with the sig- demonstrate that BW723C86 modifies the macrophage transcriptome naling capability and pathological significance of 5-HT2B (38) through both 5-HT2B–dependent (SB204741-sensitive) and 5-HT2B– (Supplemental Fig. 1), and affects the expression of genes associated independent mechanisms. to the gene ontology (GO) terms “GO heart valve development ” and “GO regulation of myeloid leukocyte differentiation” (Supplemental BW723C86 modifies the LPS-induced cytokine and gene profile Fig. 1), in line with the involvement of 5-HT2B in heart development As GSEA revealed that BW723C86 downregulates the TNF-

(28) and macrophage differentiation (26). a signaling via NFkb and inflammatory response gene sets Downloaded from To identify the genes whose expression is regulated in a 5-HT2B– (Supplemental Fig. 1), we next assessed the ability of BW723C86 dependent manner, the transcriptional effects of the 5-HT2B ago- to alter macrophage responses to an inflammatory stimulus like nist BW723C86 were also determined in the presence of LPS (Supplemental Fig. 2A). BW723C86 did not modify the basal SB204741, a selective 5-HT2B antagonist with 100-fold selectivity cytokine production by M-MØ (Supplemental Fig. 2B and data over 5-HT2C and 5-HT2A (51) that is widely used in vivo (56–58) not shown) but significantly reduced the LPS-induced production

(BW723C86 + SB204741; Fig. 1A, Supplemental Table II). We of TNF-a and increased the LPS-induced release of CCL2 http://www.jimmunol.org/ reasoned that the genes significantly modulated by BW723C86, (Supplemental Fig. 2B). A similar trend was observed with AMS, a but not by BW723C86 + SB204741, would represent bona fide less selective 5-HT2B agonist (43), although its effects did not reach 5-HT2B–regulated genes. In the presence of the antagonist statistical significance (data not shown). Importantly, the effect of SB204741, BW723C86 significantly (adj p , 0.002) increased the BW723C86 on cytokine production by LPS-activated M-MØ was expression of 155 annotated genes and downregulated the ex- blunted by the 5-HT2B antagonist SB204741 (Supplemental Fig. 2B), pression of 70 genes (Fig. 1B, Supplemental Table II). Compari- implying that BW723C86 modifies the LPS-induced cytokine profile son of the BW723C86-induced transcriptional changes in the via 5-HT2B engagement. In fact, BW723C86 significantly impaired by guest on September 29, 2021

FIGURE 1. The 5-HT2B agonist BW723C86 (BW) modifies the gene signature of human M-MØ through 5-HT2B–dependent and –independent mechanisms. (A) Experimental design of the gene-profiling experiments. (B) Number of annotated genes whose expression is significantly (adj p , 0.002) upregulated (UP) or downregulated (DOWN) in M-MØ after a 6-h exposure to BW in the absence (BW versus DMSO) or presence of SB204741 (SB) (BW + SB versus SB). (C) Number of SB-sensitive and SB-insensitive annotated genes within the group of genes significantly (adj p , 0.002) regulated by BW (genes UP by BW, left panel; genes DOWN by BW, right panel). (D) Volcano plot representation of the microarray data. Gene expression profiles of

BW-treated M-MØ versus control M-MØ (left panel) and SB + BW–treated M-MØ versus SB-treated M-MØ (right panel) are plotted according to the log2 fold change (x-axis) and log10 adj p value (y-axis). In both plots, the shaded area includes the genes with adj p , 0.05. Some of the differentially expressed genes in each case are indicated (note the distinct relative position of CYP1B1 and AHRR in both plots). (E) Heatmap of the relative expression of the 217 genes, whose significant (adj p , 0.002) upregulation by BW is prevented in the presence of the SB antagonist (SB + BW). The identity of some of the genes included within this group are indicated. 4 5-HT2B SHAPES THE MACROPHAGE TRANSCRIPTOME VIA AhR the acquisition of the characteristic transcriptome of LPS-stimulated AhR target genes like CYP1B1, AHRR, and SEMA6B was im- M-MØ (GSE99056) (59), an effect that was also abolished by the paired in M-MØ that had been previously exposed to the 5-HT2B 5-HT2B antagonist SB204741 (Supplemental Fig. 2C). Thus, en- antagonist SB204741 (Fig. 2B, Supplemental Table II). gagement of 5-HT2B by BW723C86 conditions human macrophages Based on the above findings, we assessed the ability of BW723C86 for altered responses to LPS. to upregulate the expression of xenobiotic response genes in independent validation samples and found that BW723C86 increased BW723C86 upregulates the expression of AhR target genes the expression of AhR target genes (AHRR, CYP1B1, SERPINB2, via 5-HT 2B TIPARP, EREG) in M-MØ (Fig. 2C). The upregulation of AhR Further analysis of the transcriptome of BW723C86-treated M-MØ target genes was detected as early as 2 h after addition of BW723C86 by GSEA and Enrichr (http://amp.pharm.mssm.edu/Enrichr/) (Fig. 2D). More importantly, and in agreement with microarray data, revealed a positive enrichment of genes associated with the terms the upregulation of AhR target genes by BW723C86 was signifi- metabolism of xenobiotics by cytochrome P450 gene set cantly impaired by the 5-HT2B antagonist SB204741 (Fig. 2E). Al- (Supplemental Fig. 1), AhR pathway_homosapiens_WP2873, and together, these results demonstrated that BW723C86 upregulates the AhR_homosapiens_WP2586 gene sets (data not shown), sug- expression of AhR target genes in human macrophages in a 5-HT2B– gesting that BW723C86 might influence the expression of genes dependent manner. regulated by the AhR (60), the major regulator of the biological responses to xenobiotics (61). Indeed, the 217 genes upregulated The 5-HT2B agonist BW723C86 enhances the transcriptional activity of AhR in M-MØ by BW723C86 in a 5-HT2B–dependent manner included AhR target genes like CYP1B1 and AHRR (Fig. 1E). Further, GSEA Dose response analysis revealed that the 5-HT2B agonist BW723C86 Downloaded from evidenced that exposure of M-MØ to the 5-HT2B agonist promotes the upregulation of AhR target genes in M-MØ at con- BW723C86 leads to enhanced expression of genes upregulated by centrations ranging between 0.1 and 10 mM (Fig. 3A). Thus, the AhR agonists benzopyrene (in human macrophages) and VAF- we next evaluated whether exposure to BW723C86 leads to AhR 347 (in human dendritic cells) (60, 62) (Fig. 2A, upper panels) and transcriptional activation in M-MØ. To that end, the transcriptional that this positive enrichment was prevented by the 5-HT2B an- effects of BW723C86 were determined after either pharmacological

tagonist SB204741 (Fig. 2A, lower panels). In line with GSEA or small interfering RNA–mediated inhibition of AhR in M-MØ. http://www.jimmunol.org/ results, the BW723C86-mediated upregulation of paradigmatic Pretreatment with the AhR antagonist CH223191 (63) prevented the by guest on September 29, 2021

FIGURE 2. BW723C86 upregulates the expression of AhR target genes in a 5-HT2B–dependent manner. (A) GSEA (http://software.broadinstitute.org/ gsea/index.jsp) (50) analysis of the statistic-ranked list of genes obtained from the BW723C86-treated M-MØ versus control M-MØ (upper panels) and BW723C86 + SB204741–treated M-MØ versus SB204741-treated M-MØ (lower panels) limma analysis, using data sets containing the genes upregulated by benzopyrene in human macrophages (AHR_UP_BPA_8H) or VAF-347 in human dendritic cells (DCs) (VAF347_UP in DC logFC .1) (60, 62). (B) Heatmap of the expression of four representative AhR target genes in the transcriptome of BW723C86-treated M-MØ, SB204741-treated M-MØ, and SB204741 + BW723C86–treated M-MØ. (C) Relative expression of the indicated genes in nontreated (2) and BW723C86-treated M-MØ (6 h) (+) (n =7–11).*p , 0.05, **p , 0.01, ***p , 0.001. (D) Relative expression of CYP1B1 and TIPARP in nontreated (2) and M-MØ treated with BW723C86 (+) for the indicated periods of time (n =3).*p , 0.05, **p , 0.01, ***p , 0.001. (E) Relative expression of CYP1B1, AHRR,andTIPARP in nontreated (2)or M-MØ treated for 6 h with BW723C86, SB204741, or both (+) (n =5).*p , 0.05, **p , 0.01, ***p , 0.001. (C–E) Results are shown as the expression of each gene after the different treatments and relative to its expression in control (untreated) samples. In all cases, mean and SEM are shown. The Journal of Immunology 5 Downloaded from

FIGURE 3. CYP1B1 upregulation by BW723C86 (BW) is dependent on 5-HT2B and requires AhR activation. (A) Relative expression of CYP1B1 in nontreated (0) or M-MØ treated for 6 h with the indicated concentrations of BW (n = 3). *p , 0.05. (B) Relative expression of CYP1B1 and TIPARP in M-MØ either untreated (2) or treated for 6 h with BW in the absence or presence (+) of the AhR antagonist CH223191. M-MØ were treated in parallel with the AhR agonist FICZ (6 h) for control purposes. Results are shown as the expression of each gene after the different treatments and relative to its ex- http://www.jimmunol.org/ pression in control (untreated) samples. In all cases, mean and SEM are shown (n = 4). *p , 0.05, ***p , 0.0005. (C and D) AHR mRNA (C) and AhR protein level (D) in M-MØ transfected with siC or an siAhR. Eighteen hours after transfection, M-MØ were washed and lysed for real-time quantitative RT- PCR (n = 6). ***p , 0.001 (C). Western blots from two independent knockdown experiments are shown in (D). (E) Relative expression of CYP1B1 and TIPARP in M-MØ transfected with siC or siAhR and either left untreated (2) or treated for 6 h with BW (+) (n = 6). *p , 0.05, **p , 0.01, ***p , 0.001. In (A)–(E), results are shown as the expression of each gene after the different treatments and relative to its expression in control (untreated) samples. In all cases, mean and SEM are shown. (F) AhR-dependent transcriptional activity in XRE-luciferase–transfected M-MØ left untreated (2) or treated for 14 h with BW (+) in the absence or presence of the AhR antagonist CH223191, which was added 1 h before agonist addition. Mean and SEM of the AhR- dependent luciferase activity of ﬁve independent experiments is shown (n = 5). *p , 0.05, **p , 0.01.

BW273C86-induced upregulation of both CYP1B1 and TIPARP, 6-APB (10 nM–1 mM) upregulated CYP1B1 expression in M-MØ by guest on September 29, 2021 whose expression could be also increased by the AhR agonist FICZ (Fig. 5A). Similarly, the 5-HT2B agonist AMS enhanced macro- (64) (Fig. 3B). Besides, small interfering RNA–mediated knock- phage CYP1B1 expression in a dose-dependent manner, and its down of AhR (Fig. 3C, 3D) completely prevented the BW273C86- effect was abolished by the SB204741 antagonist (Fig. 5B). Next, induced upregulation of the AhR target genes CYP1B1 and TIPARP we also tested whether 5-HT had an impact on the BW723C86- (Fig. 3E). Further supporting the involvement of AhR in the induced transcriptional effects and found that the BW723C86- BW723C86-induced upregulation of AhR target genes, reporter mediated upregulation of CYP1B1 was reduced by 100 mM5-HT, gene experiments revealed that BW723C86 specifically en- whose presence caused a slight increase in the expression of the hances the transcriptional activity of AhR in M-MØ, an effect AhR target gene CYP1B1 (Fig. 5C). These results indicated that that was abrogated by the AhR antagonist CH223191 (Fig. 3F). 5-HT2B agonists are capable of upregulating AhR target gene To find out whether the ability of the 5-HT2B agonist BW723C86 expression in a 5-HT2B–dependent manner in human macrophages to activate AhR is cell type specific, a similar set of experiments and suggested that the presence of 5-HT interferes with the ability was performed on 5-HT2B–expressing human hepatoma HepG2 of the 5-HT2B agonist BW723C86 to upregulate AhR target genes cells. As shown in Fig. 4A, BW723C86 enhanced the expression in human macrophages. of CYP1B1 in HepG2 cells after a 4- to 6-h exposure. The 5-HT 2B Serotonin increases the expression of AhR target genes in a agonist significantly potentiated the transcriptional activity of dose-dependent manner and through interaction with 5-HT AhR in HepG2 cells, and the BW723C86-induced AhR tran- 2B scriptional activity was prevented in the presence of the 5-HT2B Given the above results, and to address whether 5-HT influences antagonist SB204741 (Fig. 4B). Moreover, the BW723C86- the expression of AhR target genes, we initially determined the induced AhR transcriptional activity in HepG2 was greatly im- transcriptome of M-MØ exposed to BW723C86, 10 mM 5-HT, or paired by the AhR antagonist CH223191 (Fig. 4C). Therefore, 100 mM 5-HT using RNAseq. The expression of the AhR target engagement of the 5-HT2B serotonin receptor by BW723C86 re- genes was variably elevated in response to each stimulus, but a sults in enhancement of the transcriptional activity of AhR and core of AhR-regulated genes (including SERPINB2, CYP1B1, upregulates the expression of AhR target genes. GPR183, SEMA6B, and IL-1B) was enhanced by all three treatments (Fig. 6A). In fact, the transcriptome of BW723C86- and Additional 5-HT2B ligands influence the expression of 5-HT–treated M-MØ exhibited a positive enrichment of the BW723C86-regulated genes benzopyrene-upregulated (Fig. 6B, upper panels) and VAF-347– To further support the involvement of 5-HT2B in the ability of upregulated gene sets (60, 62) (Fig. 6B, lower panels) by GSEA, BW723C86 to promote AhR activation, the influence of other whose respective leading edges shared AhR-regulated genes 5-HT2B agonists on AhR target gene expression was determined. (Fig. 6C). Therefore, we evaluated the effect of 5-HT (100 mM) on Albeit to a lower extent than BW723C86, the 5-HT2B agonist the expression of representative AhR targets in the presence of an 6 5-HT2B SHAPES THE MACROPHAGE TRANSCRIPTOME VIA AhR

FIGURE 4. BW723C86 also promotes CYP1B1 upregulation and AhR activation in HepG2 cells via 5-HT2B.(A) Relative expression of CYP1B1 in nontreated (2) or BW723C86-treated (+) HepG2 hepatoma cells exposed to the agonist for the indicated periods of time (n =3)*p , 0.05, **p , 0.01. Results are shown as CYP1B1 expression in BW723C86-treated cells relative to its expression in untreated cells. In all cases, mean and SEM are shown. (B) AhR-dependent transcriptional activity in XRE-luciferase–transfected HepG2 cells left untreated (2) or treated with BW723C86 (16 h) in the absence or presence of the indicated concentrations of the 5-HT2B antagonist SB204741, which was added 1 h before the agonist (n = 4). ***p , 0.001. (C) AhR- dependent transcriptional activity in XRE-luciferase–transfected HepG2 cells left untreated (2) or treated with BW723C86 (16 h) in the absence or n presence of the AhR antagonist CH223191, which was added 1 h before agonist addition. HepG2 cells were treated with FICZ for control purposes ( = 4). Downloaded from ***p , 0.001. In (B) and (C), results are shown as the mean and SEM of the relative AhR-dependent luciferase activity of each sample.

inhibitor of AhR (CH223191) or antagonists of either 5-HT2B expression was reduced at high 5-HT concentrations. The in- (SB204741) or 5-HT7 (SB269970) (Fig. 6D). Although 5-HT involvement of 5-HT2B in the transcriptional effects of high 5-HT creased the expression of all assayed genes (Fig. 6E), the presence concentrations was confirmed through the use of the 5-HT2B an- of the AhR inhibitor significantly reduced the 5-HT–mediated tagonist SB204741, which significantly impaired the 5-HT– http://www.jimmunol.org/ upregulation of CYP1B1, SEMA6B, SERPINB2,andEREG but had mediated upregulation of CYP1B1, SEMA6B,andAHRR (Fig. 7B). no effect on PDE2A, whose 5-HT–mediated upregulation is de- Therefore, serotonin modulates the transcriptional profile of human pendent on the 5-HT7–protein kinase A (PKA) axis (25) (Fig. 6E). macrophages through the 5-HT2B–dependent activation of AhR. Importantly, the 5-HT–mediated upregulation of CYP1B1, SEMA6B, and SERPINB2 was impaired in the presence of the 5-HT2B antag- Discussion onist SB204741, which had no effect on the upregulation of The polarization-specific expression of 5-HT2B, an unintended PDE2A (Fig. 6F). Further, the 5-HT7 antagonist SB269970 had no target of commonly used drugs, prompted the analysis of the effect on the expression of CYP1B1 but completely prevented the transcriptional consequences of 5-HT2B activation in human mac-

5-HT–mediated upregulation of GPR183, EREG and, as expected rophages and has led to the finding that engagement of 5-HT2B by by guest on September 29, 2021 (25), PDE2A (Fig. 6G). Altogether, these results indicate that either serotonin or 5-HT2B agonists shapes the macrophage tran- serotonin regulates the expression of paradigmatic AhR target scriptome partly via activation of AhR (61). The identification of genes (CYP1B1, SEMA6B, SERPINB2) via 5-HT2B and also the 5-HT/5-HT2B/AhR axis supports a role for 5-HT2B in myeloid modulates the expression of additional AhR targets via 5-HT7. cell differentiation, extends the range of cell-specific intracellular Because the concentration of peripheral 5-HT ranges from signaling pathways initiated upon serotonin receptor engagement, nanomolar levels (in plasma) to millimolar levels (in sites of in- and illustrates a novel mechanism by which 5-HT can modulate flammation, thrombus formation, and fibrosis) (12, 65–67), we inflammatory responses. In this regard, our results indicate that next sought to determine the effects of different 5-HT concen- 5-HT2B engagement shifts the TNF/CCL2 ratio toward the anti- trations on the expression of AhR targets. As shown in Fig. 7A, inflammatory side in LPS-treated macrophages. Besides, the ability 5-HT dose dependently enhanced the expression of the AhR target of 5-HT to modify the expression of AhR targets implies its po- genes (CYP1B1, SEMA6B, AHRR) at concentrations between tential capacity to modulate cellular responses toward endoge- 10 and 500 mM, whereas its 5-HT7–independent effect on PDE2A nous and exogenous agents that trigger AhR activation.

FIGURE 5. AhR activation in human macrophages is also triggered by other 5-HT2B ligands and modulated by serotonin. (A) Relative expression of CYP1B1 in M-MØ treated for 6 h with DMSO (vehicle; 2), BW723C86 (+), or with increasing concentrations of 6-APB (10, 50, 100 nM, or 1 mM) (n = 9). (B) Relative expression of CYP1B1 in nontreated (2) or M-MØ treated for 6 h with BW723C86 (+) or with increasing concentrations of AMS (10, 20, 50 nM, 100, or 500 mM) (left panel) (n = 6) and either in the absence or presence of SB204741 using 100 or 500 mM AMS (right panel) (n = 4). (C) Relative expression of CYP1B1 in M-MØ either untreated (2) or treated for 6 h with 10 mM BW723C86, 5-HT (10 or 100 mM), or both (n = 4). (A–C) CYP1B1 expression after the different treatments and relative to its expression in control (untreated) samples is shown. Mean and SEM are shown. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Serotonin upregulates AhR target gene expression in human macrophages. (A) Heatmap of the log2 fold change of the expression of the indicated AhR-responsive genes in M-MØ exposed for 6 h to BW723C86, 10 mM 5-HT, or 100 mM 5-HT, as determined by RNAseq. (B) GSEA (50) analysis of the statistic-ranked list of genes obtained from the limma analysis of the BW723C86-treated M-MØ versus control M-MØ (left panels), 10 mM 5-HT–treated M-MØ versus control M-MØ (center panels), and 100 mM 5-HT–treated M-MØ versus control M-MØ (right panels) transcriptomes, using data sets containing the genes upregulated by benzopyrene in human macrophages (AHR_UP_BPA_8H) or VAF-347 in human dendritic cells (DCs) (VAF347_UP in DC logFC .1) (60, 62). Normalized enrichment score (NES), false discovery rate q value, and familywise error rate p values are indicated in each case. (C) Venn diagrams of the genes included within the leading edge of the GSEA panels shown (B). (D) Schematic representation of the experiments shown in (E)–(G). (E–G) Relative expression of the indicated genes in nontreated M-MØ (2) or M-MØ exposed to 100 mM 5-HT (6 h) in the absence (2) or presence of the AhR inhibitor CH223191 (E), the 5-HT2B antagonist SB204741 (F), or the 5-HT7 antagonist SB269970 (G)(n = 3). *p , 0.05, **p , 0.01. Results are shown as the expression of each gene under the different treatments. In all cases, mean and SEM are shown.

Remarkably, although 5-HT and BW723C86 share the ability to like PDE2A (25). Because PKA activation (or increased cAMP activate AhR via 5-HT2B, the transcriptional effects of the en- levels) represses AhR-dependent gene expression (70), it is gagement of 5-HT2B by BW723C86 on human macrophages are therefore possible that the PKA activation triggered upon 5-HT distinct from those triggered by 5-HT because 5-HT can engage binding to 5-HT7 might limit the activation of AhR secondary to both 5-HT2B and 5-HT7 in human M-MØ (26). Thus, although 5-HT binding to 5-HT2B. In line with this hypothesis, it is worth 5-HT2B and 5-HT7 are expressed by anti-inflammatory IL-10– noting that PDE2A gene expression in macrophages is greatly producing human macrophages (26) and promote a profibrotic upregulated after 5-HT binding to 5-HT7 (25) and that PDE2A phenotype (12, 25, 68, 69), their individual engagement has interaction with the immunophilin-like protein XAP2 (a member different transcriptional outcomes; 5-HT2B specifically promotes of the molecular complex that retains unliganded AhR in the AhR activation, whereas 5-HT7 drives the acquisition of a PKA- cytoplasm) inhibits dioxin-induced AhR nuclear translocation dependent transcriptome (25). Therefore, 5-HT appears to pref- and transcription in hepatocytes (71). erentially engage 5-HT7 over 5-HT2B because the transcriptome A link between 5-HT and AhR has been recently proposed in the of 5-HT–treated macrophages at early time points (6 h) is pre- case of intestinal epithelial cells, in which 5-HT activates AhR dominantly shaped by 5-HT7–initiated signals (25). Interest- independently on 5-HT receptors and via the serotonin transporter ingly, ligation of 5-HT7 by 5-HT triggers PKA-dependent (SERT) (72). The BW723C86- and 5-HT–mediated activation of signaling and transcription in macrophages (25) and other cell AhR in macrophages that we now describe is unrelated to the types (37) and results in the expression of PKA-regulated genes 5-HT/SERT/AhR link because it is sensitive to the presence of an 8 5-HT2B SHAPES THE MACROPHAGE TRANSCRIPTOME VIA AhR

FIGURE 7. Serotonin upregulates AhR target gene expression in human macrophages in a dose-dependent manner. (A) Relative expression of the indicated genes in nontreated M-MØ (2)orM-MØ exposedfor6htoBW723C86orin- creasing amounts of 5-HT (10, 100, 500 mM). (B) Relative expression of the indicated genes in nontreated M-MØ (2) or M-MØ exposed for 6 h to 500 mM 5-HT in the absence (2)orpresenceof the 5-HT2B antagonist SB204741. Results are shown as the expression of each gene after the different treatments, and mean and SEM are shown (n =3).*p , 0.05, , , **p 0.01, ***p 0.001. Downloaded from

5-HT2B antagonist and because M-MØ are devoid of SERT expres- of multinucleated cells during M-CSF + RANKL–driven osteo- http://www.jimmunol.org/ sion (73). The existence of these two pathways (5-HT/SERT/AhR clastogenesis (data not shown). Therefore, BW723C86 impairs in intestinal epithelialcellsand5-HT/5-HT2B/AhR in macro- osteoclast differentiation in a 5-HT2B–independent manner, a phages) fits with the AhR-activating ability of tryptophan ca- previously unnoticed action of this 5-HT2B agonist that should tabolites (74) and supports the importance of 5-HT and related be considered when evaluating its effects in vivo. molecules in modulating inflammatory responses. Thus, and besides a receptor for environmental toxins (61), AhR mediates Disclosures vascular system development, immune system polarization, and The authors have no financial conflicts of interest. resolution of inflammatory responses (75, 76), processes in which 5-HT has been also implicated (9, 38, 39, 77). 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FDRq 0.10 FDRq 0.078 FDRq 0.088

Supplemental Figure 1. Gene Ontology analysis of the BW723C86-treated M-MØ transcriptome. Selected GSEA (http://software.broadinstitute.org/gsea/index.jsp) on the “t statistic-ranked” list of genes obtained from the BW723C86-treated M-MØ versus untreated-M-MØ limma analysis, using the available data sets. The FDRq value is indicated in each case. M-MØ BW723C86 LPS A Cytokine production 1h 6h 18h Gene expression SB204741

CCL2 (ng/ml) TNF (ng/ml) * B 80 *** 12 * 10 60 8 *** 40 6 4 20 2 LPS - - - - + + + + - - - - + + + + BW723C86 - + - + - + - + - + - + - + - + SB204741 - - + + - - + + - - + + - - + +

BMP6 RGS16 CCL19 SOCS2 ARNT2

A 1.5 10 1.0 1.0 * C 15 *** *** * n *

RN 8 0.8 0.8 o i

m 1.0 10 6 0.6 0.6 ess ve r i p t 4 0.4 0.4

a 0.5

l 5 ex

e 2 0.2 0.2 R LPS ++++ ++++ ++++ ++++ ++++ BW723C86 - + - + - + - + - + - + - + - + - + - + SB204741 - - ++ - - ++ - - ++ - - ++ - - ++

Supplemental Figure 2. The 5-HT2B agonist BW723C86 conditions human macrophages for altered LPS-stimulated cytokine and gene expression. A. Experimental design to assess the effect of BW723C86 and SB204741 on LPS-stimulated cytokine production and gene expression in M-MØ. B. Production of CCL2 and TNF from LPS-stimulated (18h) M-MØ that had been non-treated (-) or exposed (6 h) to BW723C86, SB204741 or both (+), as determined by ELISA. Mean and SEM of 10/12 independent experiments are shown (*, p < 0.05; ***, p < 0.001). C. Expression of the indicated genes in LPS-stimulated (4h) M-MØ that had been non-treated (-) or exposed (6 h) to BW723C86, SB204741 or both (+), as determined by qRT-PCR. Results are expressed as the mRNA level of each gene relative to the level of TBP mRNA in the same sample. Mean and SEM of six independent experiments is shown (*, p < 0.05; ***, p < 0.001). Supplemental Table 1. Oligonucleotides used for RT-PCR GENE OLIGO SENSE OLIGO ANTISENSE BMP6 5´- acatggtcatgagctttgtga-3´ 5´- actctttgtggtgtcgctga-3´ RGS16 5´- agggtccacctgtgagca-3´ 5´- actcagagagggcacatcca-3´ CCL19 5´- gcctgctggttctctggac-3´ 5´- ggatgggtttctgggtcac-3´ SOCS2 5´- cagtcaccaagccccttc-3´ 5´- aagggatggggctctttct-3´ ARNT2 5´- tgcaggaattagtgcatcg-3´ 5´- tgggcttccttgggagtag-3´ ACSL1 5´- ggagtgggctgcagtgac-3´ 5´- gggcttgcattgtcctgt-3´ DUSP6 5´- ttggaacttactgaagccacct-3´ 5´- cgactggaacgagaatacgg-3´ EGR1 5´- agccctacgagcacctgac-3´ 5´- ggtttggctggggtaactg-3´ NCF4 5´- gcagcactcccctattgaaa-3´ 5´- cagagctatgtcctctctctgga-3´ CYP1B1 5´- acgtaccggccactatcact-3´ 5´- ctcgagtctgcacatcagga-3´ TIPARP 5´- tctcaggctcccgttcag-3´ 5´- tggtttccatttccataatgtg-3´ AHRR 5´- gcaaaacccagagcagacac-3´ 5´- acagactggtggtggctttta-3´ EREG 5´- tggtctcttcactcaggtctc-3´ 5´- cgtgagttggcatagggaac-3´ SERPINB2 5´- catggagcatctcgtccac-3´ 5´- actgcattggctcccactt-3´ OCSTAMP 5´- cacgctcacggtcaagtatg-3´ 5´- taggagctgtggacggagag-3´ CSF1 5´-gcaagaactgcaacaacagc-3´ 5´- agttcgaatcaggcttggtc-3´ DCSTAMP 5´- gcatgcaaagctgcttaaaa-3´ 5´- ggactggaagccagaaatga-3´ ACP5 5´- cggccacgatcacaatct-3´ 5´- cgctttgaggggtccatga-3´ CTSK 5´- gccagacaacagatttcc-3´ 5´-cagagcaaagctcaccac-3´ CALCR 5´- cgcttgcggtggtattatct-3´ 5´- ggtaatagcatggatagtggttgg-3´ TNFRSF11A 5´- catcatgggacagagaaatcc-3´ 5´- ggctaaacatggggttc-3´ CA2 5´- cacccctcctcttctggaat-3´ 5´- agtttacggaatttcaacacctg-3´ MITF 5´- cccctccccttctcctta-3´ 5´- cattctcctgggcttgctc-3´ MITF (A) 5´- caaaagtcaaccgctgaaga-3´ 5´- aggagcttatcggaggcttg-3´ FOS 5´- ctaccactcacccgcagact-3´ 5´- aggtccgtgcagaagtcct-3´ TRAF 6 5´- tcctctaccagcgccttg-3´ 5´- tgggtcccttcagaagttcat-3´ BCL6 5´- ttccgctacaagggcaac-3´ 5´- tgcaacgatagggtttctca-3´ PRDM1 5´- acgtgtgggtacgaccttg-3´ 5´- ctgccaatccctgaaacct-3´ IRF8 5´- cagctccttccagactggtg-3´ 5´- taggtggtgtaccccgtcac-3´ TBP 5´- cggctgtttaacttcgcttc-3´ 5´- cacacgccaagaaacagtga-3´