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LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response

Cell, Vol 119, 299-309, 15 October 2004

Sean B. Joseph,1,4 Michelle N. Bradley,1,4 Antonio Castrillo,1,4 Kevin W. Bruhn,2 Puiying A. Mak,3 Liming Pei,1 John Hogenesch,3 Ryan M. O'Connell,2 Genhong Cheng,2 Enrique Saez,3 Jeffery F. Miller,2 and Peter Tontonoz1

The nuclear receptors LXRα and LXRβ are important regulators of lipid metabolism in many cell types. LXRs bind to DNA as heterodimers with the retinoid X receptor (RXR). Physiologic activators for the LXRs include oxysterols and intermediates in the cholesterol biosynthetic pathway (Janowski et al., 1996; Lehmann et al., 1997). LXR activity controls bile acid synthesis in the liver and regulates cholesterol absorption in the intestine (Peet et al., 1998; Repa et al., 2000; Tangirala et al., 2002). In macrophages, scavenger receptor-mediated uptake of oxidized lipoproteins leads to the transcriptional activation of LXRs and the induction of genes, such as ABCA1 and apoE, that facilitate cholesterol removal from the cell (Repa and Mangelsdorf, 2000; Tontonoz and Mangelsdorf, 2003). Both LXRα and LXRβ are expressed at high levels in macrophages, and activation of either receptor promotes cholesterol efflux, indicating that the function of these receptors in macrophage lipid metabolism is largely overlapping.

Macrophages are essential for innate and adaptive immunity; however the function of LXRs in this context is not yet clear. Macrophages play a direct role in microbial killing and orchestrate inflammatory responses through the release of immune modulators such as chemokines and cytokines. The same scavenger receptors that mediate uptake of oxidized lipoproteins during atherosclerosis (e.g., SR-A, CD36) are also critical for the clearance of bacterial pathogens and apoptotic cells (Glass and Witztum, 2001; Terpstra et al., 2000). Accordingly, defects in macrophage scavenger receptor function are associated with pathological susceptibility to microbial infection (Suzuki et al., 1997; van der Laan et al., 1999). Previous studies have shown that expression of certain scavenger receptors can be modulated by nuclear receptors. For example, PPARγ positively regulates CD36 and negatively regulates SR-A (Ricote et al., 1998; Tontonoz et al., 1998). However, the significance of this regulation in the setting of microbial infection is unknown.

Recent studies have also revealed the existence of crosstalk between macrophage inflammatory pathways and nuclear receptor signaling. Synthetic ligands for several different orphan nuclear receptors, including PPARγ, PPARα, PPARδ, and LXR, have been reported to inhibit inflammatory gene expression (Joseph et al., 2003; Lee et al., 2003; Ricote et al., 1998; Ziouzenkova et al., 2003). For example, LXR ligands inhibit the LPS- or cytokine-induced expression of inflammatory genes such as iNOS and IL-6 by interfering with NF-kB signaling (Joseph et al., 2003). Other studies have shown that activation of TLR3 or TLR4 by microbial ligands inhibits the expression of LXR-dependent cholesterol efflux genes through a mechanism involving the transcription factor IRF3 (Castrillo et al., 2003). These observations have implications for the pharmaceutical control of inflammation and the pathogenesis of atherosclerosis; however, the role of such crosstalk in normal physiology remains unknown. The possibility that nuclear receptor signaling may be directly involved in antimicrobial responses has not been adequately explored. We demonstrate here that LXR-dependent gene expression impacts macrophage function in the setting of bacterial infection. These results outline an unexpected role for an orphan nuclear receptor pathway in innate immunity.

Mice Lacking LXRs Are Susceptible to Bacterial Infection
To investigate the potential involvement of LXRs in innate immunity, we challenged LXRαβ−/− mice with the gram-positive intracellular bacteria Listeria monocytogenes (LM). Infection with LM triggers an immediate innate response involving macrophages, neutrophils, and natural killer cells. This model organism has been used to define the importance of numerous proteins in immune function (Edelson and Unanue, 2000; North et al., 1997). Mice lacking expression of LXRs were highly susceptible to infection with LM (Figure 1A) . Whereas LXRαβ+/+ (WT) mice effectively cleared an i.v. dose of 1 × 104 cfu, none of the background-matched LXRαβ−/− mice survived (p < 0.0006). At higher doses of LM (1 × 105 cfu), LXRαβ−/− mice succumbed to the infection 2–3 days sooner than their WT controls (p < 0.0009). LXR-null mice also showed increased susceptibility when compared to pure strain C57bl/6 or sv129 controls (not shown). Surprisingly, susceptibility to LM was associated primarily with the loss of LXRα (Figure 1A). This LXRα-selective phenotype was unexpected since the macrophage cholesterol efflux pathway is redundantly controlled by both LXRs (Laffitte et al., 2001b).

 
Figure 1. Mice Lacking LXRs Are Susceptible to Listeria Monocytogenes Infection

(A) Survival curves of male LXRα−/−, LXRβ−/−, LXRαβ−/−, and LXRαβ+/+ (WT) mice infected with the indicate doses of LM by injection into the lateral tail vein. n = 6 per group. Statistical analysis (Wilcoxon): 1 × 105: WT versus LXRα−/− (p < 0.0017), WT versus LXRβ−/− (p < 0.025), WT versus LXRαβ−/− (p < 0.0009); 1 × 104: WT versus LXRαβ−/− (p < 0.0013); 1 × 103: WT versus LXRα−/− (p < 0.14), WT versus LXRβ−/− (p = 1.0), WT versus LXRαβ−/− (p < 0.055).

(B and C) Analysis of bacterial counts in livers 2 days after LM infection (1 × 103 cfu).

(D) Histological analysis of livers 2 days after LM infection (1 × 103 cfu). The number and size of neutrophilic abscesses characteristic of LM infection are greatly increased in LXRαβ−/− mice as revealed by H&E staining (objective magnification 5× and 40×). Many of the abscesses in LXRαβ−/− mice contain bacteria on Gram's staining (objective magnification 63×).
 

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Consistent with their survival curves, the bacterial burdens in livers of LXRαβ−/− and LXRα−/− mice on day 2 of infection were approximately 2 logs higher than in WT mice (Figures 1B and 1C), whereas counts in LXRβ−/− mice were not significantly different. Histologic analysis revealed a marked increase in size and number of neutrophilic abscesses in LXRαβ−/− mice (Figure 1D). Moreover, a high percentage of abscesses in LXR-null mice contained obvious bacteria by Gram's staining.

LXRs Are Essential for the Regulation of Gene Expression in Response to LM Infection
The rapidity of LM disease progression in LXR-null mice is consistent with a defect in innate immunity. Since LXRs are transcription factors, we reasoned that this defect must result from the loss of regulation of one or more genes critical for innate immune responses. First, we analyzed plasma expression of a panel of known inflammatory mediators in mice challenged with LM for either 5 hr or 2 days. As expected, a number of proteins were induced in response to LM infection, including IL-6, IP-10, IFNγ, IL-5, MCP-1/JE, MCP-3, MCP-5, and lymphotactin (Supplemental Figure S1 at http://www.cell.com/cgi/content/full/119/2/299/DC1/). Some cytokines, including IL-6 and IL-12p70, were significantly higher in LXRαβ-null animals, in agreement with previous work (Joseph et al., 2003). However, the concentrations of most factors were not different between genotypes. Thus, the immune defect of LXR-null mice does not appear to correlate with decreased production of any of these inflammatory mediators.

Next, we sought to identify LXR-regulated genes that might play a role in the innate immune response to LM. Our strategy was based on the hypothesis that such genes would be (1) induced during listeria infection, (2) induced by LXR agonists, (3) preferentially regulated by LXRα versus LXRβ, and (4) direct targets of LXR/RXR heterodimers. First, we performed transcriptional profiling of hepatic gene expression in mice challenged with LM. Genes showing a statistically significant difference of 2-fold or greater between WT and LXRαβ−/− mice are presented graphically in Figure 2A . As expected, LXR-null mice showed defective expression of a battery of genes, including SREBP-1c, SCD-1, and PLTP, involved in lipid metabolism (Figure 2A, controls), but these genes were not altered by LM infection.

 
Figure 2. Identification of Genes Dysregulated in LXR-Null Mice during LM Infection

(A) DNA microarray analysis of hepatic gene expression in WT and background-matched LXRαβ−/− mice (n = 6 per group) 5 hr and 2 days following infection with LM (1 × 103 cfu i.v.). Group I includes genes showing a statistically significant 2-fold or greater induction by LM infection as well as a 2-fold or greater expression in WT compared to LXRαβ−/− mice. Group II includes gene induction by LM infection showing at least 2-fold greater expression in LXRαβ−/− compared to WT mice. Control group includes selected known LXR target genes.

(B) Real-time quantitative PCR analysis of hepatic mRNA expression in WT and LXRαβ−/− mice infected with LM (n = 6 per group).

(C) Real-time PCR analysis of gene expression in liver and spleen of C57bl/6 mice treated for 3 days with 20 mg/kg GW3965 (n = 5 per group).
 

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Remarkably, we also identified two clusters of genes that were differentially regulated by LM infection in WT and LXR-null mice. The first cluster included genes that were induced in response to LM in control mice (1.5-fold or more) but deficient in LXR-null mice (Figure 2A, Group I). Of note, many of these genes encode proteins with previously defined links to immune function, and many are expressed selectively in macrophages and Kupffer cells. The most striking difference in this group was observed in the mRNA encoding SPα, also known as apoptosis inhibitor 6 (API6), and apoptosis inhibitor of macrophages (AIM), a member of the scavenger receptor cystine-rich repeat (SRCR) family (Gebe et al., 1997, 2000; Miyazaki et al., 1999). Other genes in Group I included MARCO, a second member of the SRCR family, the chemokine CCL24, the tripartite motif protein TRIM-34, and an EST of unknown function (Riken 15000001L15). The second interesting group consisted of genes that were more highly induced by LM infection (1.5-fold or more) in LXR-null mice compared to controls (Figure 2A, Group II). In this group, the largest differences were observed in the mRNAs encoding CD14 and CD68. The differential expression of these and perhaps other genes in Group II likely reflects the increased numbers of neutrophils and monocytes/macrophages present in livers of LXR-null mice (see Figure 1D). Furthermore, the increased expression of CD14 and CD68 in LXR-null mice challenged with LM strongly suggests that the reduced expression of certain macrophage genes in Group I does not reflect decreased recruitment of these cells to the liver.

Real-time quantitative PCR confirmed the differential gene expression in WT and LXR-null mice during LM infection. For example, SPα was strongly induced in response to LM infection in livers of WT mice (Figure 2B). In LXRαβ−/− mice; however, basal expression of this gene was virtually undetectable (greater than 50-fold reduction) and it was not induced by LM infection. These studies also confirmed the differential expression of MARCO, CCL24, Trim-34, Ctla2b, and Oasl11 in mice lacking LXRs.

The greater LM susceptibility of LXRα−/− mice compared to LXRβ−/− mice suggests that one or more genes preferentially controlled by LXRα are important for defense against this pathogen. In order to address whether any of the genes dysregulated in LXRαβ−/− mice were specific for LXRα, we conducted an additional profiling experiment comparing gene expression between LXRα−/− and LXRβ−/− mice infected with LM. Among the 11 genes induced by LM in control mice but not LXRαβ−/− mice (c.f. Figure 2A), only SPα, TRIM-34, and CCL24 were found to be markedly different (> 3-fold) between livers of LXRα−/− and LXRβ−/− mice infected with LM (Supplemental Figure S2A on the Cell Website). Real-time PCR analysis confirmed the differential regulation of SPα, TRIM-34, and CCL24 by LM infection in the liver of LXRα−/− and LXRβ−/− mice (Supplemental Figure S2B). To investigate the possibility that some of these genes might be directly regulated by LXRs, we examined gene expression in livers and spleens of mice treated for 3 days with the synthetic LXR agonist GW3965. Of these three genes, only SPα was consistently induced by LXR agonist treatment (Figure 2C and data not shown). LXR ligand did not promote the expression of CCL24 or TRIM-34, suggesting that their reduced expression in LXR-null mice is likely to be a secondary effect. Thus, within the limits of our approach, we were able to identify only a single gene that meets the criteria of being induced by LM, induced by synthetic LXR agonist, and differentially expressed between LXRα−/− and LXRβ−/− mice.

Transplantation of Wild-Type Bone Marrow into LXR-Null Mice Rescues the Susceptibility to LM Infection
The observation that several macrophage-specific genes are dysregulated in livers of LXRαβ−/− mice upon LM infection suggests that altered macrophage function may underlie their susceptibility. However, since LXRα is highly expressed in hepatocytes and plays the dominant role in this cell type (Peet et al., 1998), it was important to rule out a defect in hepatocyte gene expression as the basis for LM susceptibility. To address this issue, lethally irradiated WT and LXRαβ−/− recipient mice were transplanted with bone marrow from either WT or LXRαβ−/− donors (see Experimental Procedures). Twelve weeks after transplantation, mice were infected with LM (1 × 103 i.v.), and bacterial burden in the liver was determined on day 2. Transplantation of LXRαβ−/− bone marrow into WT recipients led to an increase in bacterial burden compared to mice transplanted with control WT marrow (Figure 3A) . Moreover, transplantation of WT bone marrow into LXRαβ−/− recipients completely reversed their susceptibility to LM infection. These observations point to a defect in bone marrow-derived cells as the cause of the altered innate immune response in LXR-null mice.

 
Figure 3. The Immune Defect of LXR-Null Mice Localizes to the Bone Marrow

(A) WT and LXRαβ−/− recipient mice (n = 8–12 per group) were transplanted with bone marrow from WT or LXRαβ−/− donors (see Experimental Procedures). Twelve weeks after transplantation, mice were infected with LM (1 × 103 cfu i.v.). Bacterial counts in livers were determined 2 days after infection.

(B) Real-time PCR analysis of hepatic gene expression in transplanted mice 2 days after LM infection.
 

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We also analyzed gene expression in the livers of the transplanted mice, focusing on those genes that showed selectivity for LXRα. Of these three genes, expression of SPα most closely correlated with susceptibility to LM infection (Figure 3B). Transplantation of WT bone marrow into LXRαβ−/− mice completely restored expression of SPα and also increased expression of CCL24, but expression of Trim-34 was not recovered. Transplantation of LXRαβ−/− bone marrow into WT mice decreased expression of all three genes.

Induction of LXRα Expression in Macrophages in Response to Intracellular Bacteria
Together, the above results point to altered myeloid cell function in LXR-null mice as the potential cause of their innate immune defects and suggest that LXRα plays the dominant role in this phenotype. Consistent with this hypothesis, mRNA expression of LXRα but not LXRβ was strongly induced in BMDM in response to LM (Figure 4A) . To determine whether LXRα induction was a common response to bacterial infection or specific to LM, we infected macrophages with a panel of bacteria. A similar induction was observed with the gram-negative intracellular bacteria Shigella flexneri(Figure 4B). In contrast, gram-positive extracellular (Staphlococcus aureus) or gram-negative extracellular (E. coli) bacteria were less effective inducers of LXRα expression. Thus, macrophage expression of LXRα is preferentially responsive to infection with intracellular bacteria.

 
Figure 4. A Selective Role for LXRα in Response to Intracellular Bacteria

(A) Induction of LXRα but not LXRβ mRNA expression during LM infection with intracellular bacteria. BMDM were infected with LM at an MOI of 1 and gene expression measured at the indicated times post-infection. Gene expression was measured by real-time PCR.

(B) Preferential induction of LXRα by intracellular bacteria. BMDM were infected in vitro with LM, E. coli, S. aureus, or S. flexneri as indicated.

(C) Induction of LXRα by LM is independent of the TLR pathway. BMDM from WT, TLR2−/−, TLR4−/−, and MyD88−/− mice were infected with LM as in (B).

(D) Induction of LXRα expression by activation of the NOD signaling pathway. BMDM were treated with the NOD2 ligand muramyl dipeptide (MDP 10 μg/ml [Inohara and Nunez, 2003]), LPS (100 ng/ml), or infected with LM (MOI = 1), and gene expression was determined after 4 hr.

(E) LXRα-dependent expression of SPα in macrophages. Real-time PCR analysis of gene expression in TG-elicited peritoneal macrophages from WT, LXRα−/−, LXRβ−/−, and LXRαβ−/− mice treated for 24 hr with LXR ligand (GW3965 or T1317, 1 μM) and/or RXR ligand (LG268, 100 nM).

(F) BMDM from WT, LXRα−/−, LXRβ−/−, and LXRαβ−/− mice were pretreated with vehicle or 1 μM GW3965 for 24 hr and/or infected with LM at an MOI of 1. Gene expression was analyzed 8 hr post-infection.
 

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To further explore the mechanism whereby LXRα expression is induced in response to bacterial challenge, we analyzed macrophages from mice carrying targeted mutations in TLR2 (previously shown to recognize LM), TLR4, or the key TLR adaptor molecule MyD88. Induction of LXRα mRNA by LM was preserved in each of these knockout macrophages, indicating that the effect is independent of the TLR pathway (Figure 4C). Since previous studies have shown that NOD proteins play an important role in the TLR-independent recognition of intracellular pathogens (Inohara and Nunez, 2003), we tested the ability of the NOD pathway to regulate LXRα expression. Remarkably, treatment of macrophages with the NOD2 ligand muramyl dipeptide (MDP, 10 μg/ml) induced expression of LXRα to a similar extent as infection with LM (Figure 4D). By contrast, the TLR4 ligand LPS (100 ng/ml) had only a marginal effect. These observations suggest that intracellular bacteria specifically induce macrophage expression of the LXRα gene through the NOD signaling pathway.

LXRα Controls Expression of the Scavenger Receptor Cystine-Rich Repeat Protein SPα in Macrophages
The gene expression profiling studies of Figures 3 and 4 identify SPα as a potential contributor to the innate immune defect of LXRαβ−/− mice. We therefore addressed the ability of LXRα and LXRβ to control expression of this gene in thiglycolate (TG)-elicited macrophages treated with LXR and/or RXR ligands. Consistent with the in vivo expression studies, basal and ligand-inducible expression of SPα was severely compromised in LXRα−/− but not LXRβ−/− macrophages (Figure 4E). This striking selectivity for one LXR isoform is not observed with any of the previously identified LXR target genes in macrophages, including ABCA1, ABCG1, SREBP-1c, GLUT4, PLTP, FAS, and apoE (Figure 4E and data not shown). Expression of ABCA1and SREBP-1c was comparable in cells lacking either LXRα or LXRβ alone and response to ligand was lost only in double knockout cells. In contrast to SPα, TRIM-34 and CCL24 were not regulated in response to LXR or RXR agonists in cultured macrophages (data not shown).

We further investigated expression of SPα in cultured macrophages in response to LM infection. Cells were pretreated with the LXR ligand GW3965 for 24 hr and then infected with LM for 8 hr. Interestingly, LM infection and LXR agonist had an additive effect on SPα gene expression (Figure 4F). Consistent with the in vivo studies, expression of SPα in the setting of LM infection in vitro was also dependent on LXRα. In contrast to SPα, LXR agonist inhibited the expression of the inflammatory mediator iNOS, in agreement with previous work (Joseph et al., 2003). Thus, LXRs function as both positive and negative regulators of genes involved in antimicrobial responses.

The SPα Gene Is a Direct Target for Regulation by LXRα/RXR Heterodimers
Next, we asked whether the SPα gene was a direct target of LXR/RXR heterodimers. A genome-wide bioinformatics search for potential LXREs was carried out based on the sequences of all previously identified LXREs (see Experimental Procedures). This analysis led to the identification of a putative LXRE at position −5404 in the SPα promoter (Figure 5A) . Electromobility shift assays confirmed that this element bound LXRα/RXR heterodimers with high affinity (Figure 5B). Interestingly, LXRβ/RXR heterodimers bound the SPα LXRE poorly compared with the LXRE from the GLUT4 promoter. Furthermore, a luciferase reporter driven by the SPα LXRE was preferentially activated by LXRα/RXR in transient transfection assays (Figure 5C). These data suggest that the selectivity of SPα for LXRα is achieved, at least in part, through differential interaction with the SPα promoter.

 
Figure 5. SPα Is a Selective Target Gene for LXRα/RXRα Heterodimers

(A) Alignment of LXREs from the Spα and GLUT4 promoters.

(B) Electromobility shift assay of LXRα/RXR and LXRβ/RXR heterodimer binding to LXR response elements from the GLUT4 and SPα genes. SPα LXRE but not mutant SPα LXRE (SPα mut) preferentially binds LXRα/RXR. Asterisk indicated the shifted complex in the presence of an anti-RXR antibody.

(C) Selective activation of the SPα LXRE by LXRα. HEK 293T cells were transiently transfected in quadruplicate with expression plasmids for murine LXRα or LXRβ together with luciferase reporter genes containing two copies of the GLUT4 and wild-type or mutant SPα LXRE. Cells were treated with vehicle or LXR ligand (T1317, 111 nM) as indicated.
 

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LXR Signaling Impacts Bacterial Clearance and Macrophage Survival in Response to LM Infection In Vivo
Next we investigated the consequence of loss of LXR-dependent gene expression for macrophage function during LM infection. To assess responses to infection in vivo, we elicited peritoneal macrophages from mice by peritoneal inoculation of LM. Equivalent numbers of macrophages were recovered from WT and LXR-null mice 3 days after infection (Figure 6A , left), and the numbers showed comparable expression of MHC class I and class II, CD80 and CD86 by FACS analysis, indicating that WT and LXR-null cells were appropriately activated by infection (data not shown). However, WT macrophages contained very few viable intracellular bacteria at this time point after infection, whereas LXR-deficient macrophages contained numerous intracellular bacteria (Figure 6A, right). Moreover, flow cytometry using an annexin V antibody revealed an increased rate of apoptosis in LXR-null macrophages compared to WT cells (Figure 6B). In agreement with these results, TUNEL staining showed increased apoptosis in macrophages within spleens of LXRαβ−/− mice infected with LM in vivo (Figure 6C). Furthermore, apoptosis in LXRαβ−/− spleen was ameliorated by transplantation of WT bone marrow (Figure 6D), and this effect correlated with restored expression of the antiapoptotic factor SPα (Figure 6E).

 
Figure 6. The LXR Signaling Pathway Impacts Macrophage Survival and Antimicrobial Activity during LM Infection

(A and B) Macrophages were elicited from WT and LXRαβ−/− mice by peritoneal inoculation with LM (2.5 × 103 cfu). Three days after infection, macrophages were isolated, counted, and analyzed for the presence of viable intracellular bacteria by colony-forming assays or apoptosis by FACS analysis. (A) Defective clearance of LM by LXR-null macrophages in vivo. Equivalent numbers of macrophages were recovered from each genotype (left); however, greatly increased viable bacteria were present within LXR-null cells (right). Cells were treated with gentamycin prior to lysis and colony counting in order to kill extracellular bacteria. (B) Macrophages lacking LXRs undergo accelerated apoptosis in the setting of LM infection as revealed by costaining with PI and GFP-conjugated monoclonal antibody to Annexin V. Early apoptosis (Annexin V+, PI−), late apoptosis (Annexin V+, PI+).

(C) Increased macrophage apoptosis in spleens of LXR-null mice infected with LM. Mice were infected i.p. with 1 × 103 cfu LM. Two days after infection, spleens were analyzed by TUNEL staining. Objective magnification is 10×.

(D) Transplantation of WT bone marrow into LXRαβ−/− recipient mice corrects the apoptosis defect. Mice were infected with LM as in (C) and analyzed by TUNEL and DAPI staining. Objective magnification is 10×.

(E) Correction of the apoptosis defect in LXRαβ−/− recipient mice correlates with restored expression of SPα in spleen.

(F) Increased apoptosis in LXR-null macrophages infected with LM in vitro. TG-elicted macrophages from WT and LXRαβ−/− mice were infected in vitro with LM at an MOI of 1. Six hours after infection, cells were analyzed for apoptosis by DAPI and TUNEL staining. The percentage of cells staining for both DAPI and TUNEL was determined by manually counting 400 DAPI+ cells in each genotype. Objective magnification is 10×.
 

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To address whether the increased apoptosis observed in LXRαβ−/− mice was the result of direct interaction between LM and the macrophages, we infected TG-elicited peritoneal macrophages with LM in vitro. After 6 hr of infection, macrophages lacking LXRs exhibited greatly increased apoptosis compared to WT cells (27% TUNEL/DAPI dual positive compared to 5%, Figure 6F). By contrast, there was no difference in the very low level of apoptosis observed when WT and LXR-null macrophages were cultured in the absence of LM (not shown), indicating that the increased cell death is a specific response to LM infection. These data establish that LXR activity promotes macrophage survival in the setting of LM infection and is important for effective clearance of LM in vivo.

Expression of LXRα or SPα Promotes Macrophage Survival in the Setting of LM Infection
The gene expression profiling studies presented above identified SPα as the only gene represented on our cDNA arrays that was induced in response to LM infection, preferentially regulated by LXRα, and directly induced by ligand-activated LXRα/RXR. Interestingly, previous studies have implicated SPα in the control of macrophage survival, although a link to LM infection has not been established. To directly address the role of this factor in macrophage response to LM, we performed gain-of-function experiments in RAW264.7 macrophages, which express LXRβ but not LXRα. Expression of ABCA1 was strongly induced by synthetic ligands in control RAW-vector cells, presumably through the action of LXRβ (Figure 7A) . By contrast, SPα expression was undetectable in these cells, even in the presence of LXR and RXR ligands. Forced expression of LXRα in RAW macrophages not only conferred the ability to regulate SPα (Figure 7A) but also inhibited apoptosis when these cells were challenged with LM (Figure 7B). Furthermore, TUNEL staining and caspase-3 activity assays showed that expression of SPα alone promoted cell survival in the setting of LM infection (Figures 7C and 7D), consistent with the hypothesis that SPα is the mediator of the LXRα antiapoptotic effect. Finally, both LXRα and SPα were found to promote antimicrobial activity when expressed in RAW macrophages. Quantification of viable intracellular bacteria revealed decreased numbers of LM in cells expressing either LXRα or SPα compared to vector controls (Figure 7E). Together, these results indicate that the LXR signaling pathway plays an important role in macrophage survival and antimicrobial function in the setting of LM infection and that this function is accomplished, at least in part, through LXRα-dependent regulation of SPα.

 
Figure 7. Ectopic Expression of LXRα or SPα Promotes Macrophage Survival and Antimicrobial Activity in the Setting of LM Infection

(A) Expression of LXRα in RAW 264.7 macrophages facilitates induction of SPα by LXR (GW3965, 1 μM) and RXR ligand (LG268, 100 nM). RNA expression was determined by Northern blotting.

(B) Expression of LXRα in RAW macrophages inhibits apoptosis triggered by LM infection. Caspase-3 activity was determined after infection of RAW-vector and RAW-LXRα cells with LM at an MOI of 1.

(C) Stable expression of SPα expression in RAW macrophages. SPα mRNA expression level in WT and LXRαβ−/− TG-elicited peritoneal macrophages, RAW-vector, and RAW-SPα stable cell lines was determined by Northern analysis. 36B4 was used as a control for loading and integrity of RNA.

(D) Expression of SPα in RAW macrophages inhibits apoptosis triggered by LM infection. Apoptosis was determined by caspase-3 activity (2 hr) and TUNEL staining (6 hr) after infection of RAW-vector, RAW-SPα, and RAW-LXRα cells with LM. RAW-LXRα cells were also treated with 1 μM GW3965. The percentage of cells staining for both DAPI and TUNEL was determined by manually counting 400 DAPI+ cells in each cell line. Data represent the average of triplicate experiments.

(E) Expression of LXRα or SPα inhibits intracellular bacterial infection of RAW macrophages. Duplicate plates of RAW-vector, RAW-SPα, and RAW-LXRα cells were infected with LM at an MOI of 1. RAW-LXRα cells were also treated with 1 μM GW3965. Viable intracellular bacteria were quantified by colony-forming assays at the indicated times post-infection.
 

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Discussion

Although the importance of orphan nuclear receptors in lipid metabolism is well documented, RXR heterodimers have not previously been recognized to be involved in antimicrobial responses. Potent synthetic ligands for several receptors, including the PPARs and LXRs, have been reported to inhibit inflammatory gene expression; however, the physiological relevance of these effects has not been established. We have shown here that the LXR signaling pathway is directly involved in the control of gene expression in the setting of LM infection and impacts both macrophage survival and pathogen clearance. These results establish that a common nuclear receptor pathway is important for both lipid homeostasis and innate immunity. They further suggest that the ability of LXR to modulate inflammatory gene expression, and the ability of TLRs to modulate LXR signaling, may relate to physiologic roles for LXRs in macrophage innate immune function.

Previous work has shown that LXRs are transcriptional regulators of a battery of genes involved in cholesterol and fatty acid metabolism (Repa and Mangelsdorf, 2000). Our results establish that LXRs are also important for the proper control of genes involved in the innate immune response to bacterial pathogens. Intriguingly, the susceptibility of LXR-null mice to bacterial infection results primarily from the loss of LXRα. Although LXRα and LXRβ play redundant roles in cholesterol efflux, our results point to a unique role for LXRα in immunity. Macrophage expression of LXRα is specifically induced in response to intracellular bacterial pathogens such as LM and Shigella, and the transcriptional response to infection is particularly dependent on this receptor. We used transcriptional profiling studies to identify multiple genes whose expression was altered in LXR-null mice during infection with the intracellular pathogen LM. Our strategy for paring down this list of candidate genes relied on the observation that LXRα−/− mice are more susceptible to LM than LXRβ−/− mice. Within the limits of our assays, we were able to identify only a single direct LXR target gene, SPα (also known as API6, AIM, and CD5L), that was induced during LM infection and differentially regulated by LXRα and LXRβ. However, the possibility that other genes also contribute to the immune defects of LXR-null mice cannot be excluded. In fact, the observation that mice lacking both LXRs are still more susceptible than LXRα−/− mice is consistent with the involvement of additional LXR target genes. It is also possible that LXR-dependent genes whose expression is not altered by infection, such as those involved in cholesterol and fatty acid metabolism, also contribute to the immune phenotype.

To our knowledge, SPα is the first LXR target gene to show a significant preference for one LXR isoform in macrophages. Although lipogenic gene expression in the liver is primarily controlled by LXRα, this most likely reflects the predominance of LXRα expression in hepatocytes (Repa and Mangelsdorf, 2000). In cells that express comparable levels of the two LXRs, such as macrophages, all previously identified target genes are induced by both receptors. In contrast to other known LXR target genes, SPα is not known to be involved in lipid metabolism. Rather, the SPα protein is a member of the family of SRCR proteins that has been shown to be important for macrophage/Kupffer cell immune function. Other well-characterized members of this family include scavenger receptor A, MARCO, and CD163. SPα is a negative regulator of apoptosis in macrophages and has been implicated in the regulation of immune responses during bacterial infection (Haruta et al., 2001; Kuwata et al., 2003; Miyazaki et al., 1999). A direct link between SPα and the immune response to LM, however, has not been reported previously. We have shown here that expression of SPα in macrophages promotes both macrophage survival and antimicrobial activity during LM infection. These observations strongly suggest that the enhanced susceptibility of LXR-null mice results, at least in part, from loss of regulation of macrophage SPα expression during infection. However, since LXRα is widely expressed in cells of the myeloid lineage, the possibility that cell types in addition to macrophages contribute to the phenotype should not be excluded. In particular, neutrophils are known to play a prominent role in the innate response to LM infection. Although we have not explored the function of LXRs in neutrophils in the present study, it is likely that the actions of LXRα and SPα in this cell type may also be relevant for innate immunity.

A number of studies have documented the importance of proper control of apoptosis for innate immunity. On one hand, immune cell apoptosis is likely to be an important mechanism for downregulation of inflammatory responses and the resolution of inflammation. On the other hand, premature induction of apoptosis by phagocytosed pathogens is postulated to be a key microbial strategy to evade the host immune response. A number of bacteria have been shown to directly trigger apoptosis in monocytes and macrophages, including LM, Shigella flexneri, Mycobacterium tuberculosis, and Streptococcus pneumoniae(Fettucciari et al., 2000; Hilbi et al., 1997; Rojas et al., 1997). LM has also been shown to cause apoptosis of dendritic cells through the action of hemolysin (Guzman et al., 1996). Recent studies by some of the authors (R.M.O., K.W.B., J.F.M., and G.C.) have correlated decreased macrophage apoptosis with resistance to LM infection (O'Connell et al., 2004). Thus, the ability of SPα to inhibit macrophage apoptosis triggered by LM represents a plausible mechanism to explain the enhanced antimicrobial activity of SPα-expressing cells. However, it is also possible that this SRCR protein has antimicrobial functions independent of its ability to inhibit apoptosis. Further studies will be required to better define the mechanism of action of SPα in the innate immune response.

Together with previous work, these results position LXR at the crossroads of lipid metabolism and innate immunity in macrophages. The LXR pathway not only engages in crosstalk with the NF-κB and TLR signaling pathways but also is required for the expression of genes directly involved in antimicrobial responses. Clearly, the impact of LXR signaling on macrophage gene expression is complex and context dependent. LXRs are negative regulators of many NF-κB-dependent inflammatory genes such as iNOS and IL-6, yet they are positive regulators of the SRCR family member SPα and perhaps other genes yet to be identified. Given the established role of LXRs in the efflux of lipoprotein-derived cholesterol in macrophages, these observations suggest that common nuclear receptor transcriptional pathways may be utilized to facilitate the clearance of oxidatively modified lipoproteins, apoptotic cells, and bacterial pathogens.

At present, it is not clear whether the functions of LXR in cholesterol metabolism and immunity are related or distinct. For example, it is possible that SPα is also important for macrophage survival in the setting of hypercholesterolemia or is involved in the phagocytic response to cholesterol-rich lipoproteins. It is also possible that the proper control of cholesterol metabolism is itself important for antimicrobial functions. An important goal of future investigation will be to define the role of specific LXR target genes in the macrophage response to these various physiological and pathological stimuli.

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Castrillo, A.X Joseph, S.B.X Vaidya, S.A.X Haberland, M.X Fogelman, A.M.X Cheng, G. and Tontonoz, P. (2003). Crosstalk between LXR and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism. Mol. Cell 12, 805-816. [Medline] [Summary] [Full Text]