Retinoid X Receptor Α Controls Innate Inflammatory Responses Through The

Retinoid X receptor α controls innate inflammatory responses through the up-regulation of chemokine expression Vanessa Núñeza,1, Daniel Alamedaa,1, Daniel Ricoa,2, Rubén Motab, Pilar Gonzalob, Marta Cedenillaa, Thierry Fischerc, Lisardo Boscád, Christopher K. Glasse, Alicia G. Arroyob, and Mercedes Ricotea,3 Departments of aRegenerative Cardiology and bVascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares, Madrid 28029, Spain; cDepartment of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid 28049, Spain; dInstituto de Investigaciones Biomédicas “Alberto Sols” (CSIC-UAM), Madrid 28029, Spain; and eDepartment of Medicine, Department of Cellular and Molecular Medicine, University of California, La Jolla, CA 92093 Edited* by Daniel Steinberg, University of California, La Jolla, CA, and approved April 19, 2010 (received for review November 25, 2009) The retinoid X receptor α (RXRα) plays a central role in the regulation Chemokines and their receptors have been implicated in the of many intracellular receptor signaling pathways and can mediate modulation of leukocyte trafficking, immune/inflammatory respon- ligand-dependent transcription by forming homodimers or hetero- ses, sepsis, and multiorgan failure (18, 19). Clinical studies have also dimers with other nuclear receptors. Although several members of identified elevated levels of chemokines associated with human the nuclear hormone receptor superfamily have emerged as impor- sepsis and acute lung injury (20). tant regulators of macrophage gene expression, the existence in vivo We have examined the role of RXRα in the innate immune of an RXR signaling pathway in macrophages has not been estab- system by conditionally disrupting RXRα in myeloid cells. We lished. Here, we provide evidence that RXRα regulates the transcrip- show that chemokines Ccl6 and Ccl9 are novel target genes for tion of the chemokines Ccl6 and Ccl9 in macrophages independently RXRα in primary peritoneal macrophages. RXRα deletion also of heterodimeric partners. Mice lacking RXRα in myeloid cells exhibit results in decreased levels of CCL6 and CCL9 in vivo, correlating reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes with impaired leukocyte recruitment to inflammatory sites and to sites of inflammation, and lower susceptibility to sepsis. These prolonged survival in sepsis induced by cecal ligation and puncture studies demonstrate that macrophage RXRα plays key roles in the (CLP) or lipopolysaccharide (LPS). These results establish that regulation of innate immunity and represents a potential target for RXR is involved in the regulation of the innate immune response immunotherapy of sepsis. and provide evidence for the existence of RXR signaling in macrophages in vivo. nuclear hormone receptors | macrophages | innate immunity | sepsis Results uclear receptors are ligand-dependent transcription factors RXRα Controls Chemokine Gene Expression in Macrophages. To in- α fl that regulate diverse aspects of development and homeostasis vestigate the role of RXR in in ammation and in macrophage N α fi (1, 2). Several members of the nuclear receptor superfamily have function, we generated mice lacking RXR speci cally in myeloid fl cells (RXRα KO) (Fig. S1 A–D). Gene expression profiling of WT emerged recently as key regulators of in ammation and immune α responses (2, 3). Retinoid X Receptors (RXRs) occupy a central and RXR KO peritoneal macrophages showed that Ccl6 (C10, Mrp-1, Scya6) and Ccl9 (Mip-1γ, Mrp-2, Scya9) are down-regulated position in the nuclear receptor superfamily because they form in the KO mice. Reduction in Ccl6 and Ccl9 gene expression in heterodimers with many other family members and hence are RXRα KO macrophages was confirmed by real-time quantitative involved in the control of a variety of physiologic processes (4, 5). PCR (Q-PCR) (Fig. 1A). Q-PCR further showed that the RXR RXRs are also able to activate transcription from cognate reporter α ligands 9-cis-retinoic acid (RA) and LG100268 induced Ccl6 and genes as homodimers (6, 7). There are three RXR isotypes, RXR Ccl9 gene expression in WT macrophages, but expression was not β γ (NR2B1), RXR (NR2B2), and RXR (NR2B3), which show induced by LG100754 (Fig. 1B). LG100268 and 9-cis-RA are RXR fi tissue-speci c differences in expression (4, 8). Previous studies pan-agonists, whereas LG100754 is an agonist of PPAR/RXR and suggest that the most abundant RXR in myeloid cells, or at least RAR/RXR heterodimers but an antagonist of RXR homodimers the most functionally important, is RXRα (9). RXRs are receptors (12). Ccl6 and Ccl9 mRNA levels were maximally induced after for ligands such as 9-cis-retinoic acid and endogenous fatty acids 24 h stimulation with 9-cis-RA (Fig. S2A), and the induction of (10, 11), and for a variety of synthetic agonists (called rexinoids), Ccl6 and Ccl9 by LG100268 and 9-cis-RA was inhibited by such as LG100268 (12). Selective RXR ligands are being de- LG100754 (Fig. S2B). The effect of RXR ligands on protein ex- veloped for cancer therapy and are promising agents for the pression was examined by ELISA. Treatment of macrophages with treatment of metabolic diseases (13). 9-cis-RA or LG100268 significantly increased CCL6 and CCL9 Innate immunity is an ancient form of host defense that is acti- vated rapidly to enable, through a multiplicity of effector mecha- nisms, defense against a broad spectrum of foreign substances (14). Author contributions: T.F., L.B., C.K.G., A.G.A., and M.R. designed research; V.N., D.A., Inflammation, one of the first responses of the immune system to D.R., R.M., P.G., and M.C. performed research; T.F. contributed new reagents/analytic tools; V.N., D.A., D.R., R.M., P.G., T.F., L.B., A.G.A., and M.R. analyzed data; and C.K.G., infection, is mediated by immune system cells, whose accumulation A.G.A., and M.R. wrote the paper. in injured tissues triggers the removal of the foreign agent, prevents The authors declare no conflict of interest. subsequent infections, and promotes tissue repair (15). Normally, *This Direct Submission article had a prearranged editor. fl in ammation is self-controlled in intensity and duration (16). How- 1V.N. and D.A. contributed equally to this work. ever, when this process is dysregulated, as in sepsis, excessive 2Present address: Structural Biology and Biocomputing Programme, Centro Nacional de proinflammatory mediators are released into the bloodstream, re- Investigaciones Oncológicas, Madrid 28029, Spain. sulting in multiple organ failure. Sepsis-induced multiorgan failure 3To whom correspondence should be addressed. E-mail: [email protected]. has a high death rate in humans and is one of the leading causes of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. death in intensive care units (17). 1073/pnas.0913545107/-/DCSupplemental. 10626–10631 | PNAS | June 8, 2010 | vol. 107 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.0913545107 Downloaded by guest on September 25, 2021 A B C D Of the 49 nuclear receptors encoded by the mouse genome, 8 4 8 WT systematic quantitative PCR analysis has demonstrated that 28 are α 1.0 7 RXR KO γ δ 6 * 3 * * 6 * expressed in macrophages (21). Of these, PPAR , PPAR , and * pression * 5 Nurr1 have been demonstrated to form heterodimers with RXRs * 4 2 4 CCL6 CCL6 CCL6 0.5 * 3 on DR-1 elements. RXR also forms heterodimers with LXRs, 1 2 * 2 CCL6 (ng/ml) 1 TRs, RARs, and VDR on DR-4, DR-2/5, and DR-3 elements, re- Relative Expression Relative ex Relative Relative Expression 0.0 0 0 0 8 A spectively (4). To investigate whether RXR induces Ccl6 and Ccl9 trl 8 4 trl A 6 54 trl WT KO C 5 C -R 7 C -R α 26 is G G7 c LG268 cis-RAL L cis LG2 LG transcription as a homodimer or by forming a heterodimer, we tested 9 9 9 RXR the ability of potential heterodimeric partners to influence expression 1.25 4 5 WT of these genes in macrophages. Expression of Ccl6 and Ccl9 was up- 1.00 * 2 RXRα KO 3 * * 4 * * regulated in cells treated with the RXR ligand LG100268 but not the 0.75 3 * 2 PPAR ligands rosiglitazone, GW327647, or GW610742; moreover, CCL9 CCL9 1 0.50 CCL9 2 * 1 CCL9 (ng/ml) we observed no additive or synergistic effect when PPAR and RXR 0.25 * 1 Relative Expression Relative Expression Relative Expression * 0.00 0 0 0 ligands were both present (Fig. 2H). PPAR ligands were, however, rl A l O A 54 tr WT K Ctrl -R Ct -R C able to induce the expression of the PPAR- and RXR-target genes α is G268G754 s G268G7 s-RA c L L L L ci LG268 9 9 ci 9 RXR Abcg1 and Adrp (Fig. S2D). Furthermore, no effect was observed upon treatment with the Nurr1/RXR selective ligand XCT0135980, α Fig. 1. Loss of RXR in macrophages reduces Ccl6 and Ccl9 chemokine ex- the LXR ligand T1317, the RAR ligand TTNPB, the VDR ligand Vit pression. (A) Q-PCR analysis of Ccl6 and Ccl9 mRNA expression in RXRα KO fi ≤ D3, or the TR ligand T3 (Fig. S2E). Collectively, these ndings peritoneal macrophages. *, P 0.05 compared with WT. (B) Q-PCR analysis of α Ccl6 and Ccl9 gene expression in peritoneal macrophages treated for 24 h provide evidence for an RXR signaling pathway in macrophages with the RXR ligands 9-cis-RA (1 μM), LG268 (50 nM), or LG754 (1 μM). *, P ≤ that is independent of RXR heterodimeric partners. 0.05 compared with control (Ctrl). (C) ELISA of CCL6 and CCL9 protein in macrophages treated as in C for 48 h. *, P ≤ 0.05 compared with control (Ctrl). Decreased CCL6 and CCL9 Expression Impairs Leukocyte Recruitment (D) Q-PCR analysis of Ccl6 and Ccl9 mRNA expression in WT and RXRα KO in Myeloid RXRα Knockout Mice.

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