Reprogramming of the LXRα Transcriptome Sustains Macrophage Secondary Inflammatory Responses

Abstract Macrophages regulate essential aspects of innate immunity against pathogens. In response to microbial components, macrophages activate primary and secondary inflammatory gene programs crucial for host defense. The liver X receptors (LXRα, LXRβ) are ligand‐dependent nuclear receptors that direct gene expression important for cholesterol metabolism and inflammation, but little is known about the individual roles of LXRα and LXRβ in antimicrobial responses. Here, the results demonstrate that induction of LXRα transcription by prolonged exposure to lipopolysaccharide (LPS) supports inflammatory gene expression in macrophages. LXRα transcription is induced by NF‐κB and type‐I interferon downstream of TLR4 activation. Moreover, LPS triggers a reprogramming of the LXRα cistrome that promotes cytokine and chemokine gene expression through direct LXRα binding to DNA consensus sequences within cis‐regulatory regions including enhancers. LXRα‐deficient macrophages present fewer binding of p65 NF‐κB and reduced histone H3K27 acetylation at enhancers of secondary inflammatory response genes. Mice lacking LXRα in the hematopoietic compartment show impaired responses to bacterial endotoxin in peritonitis models, exhibiting reduced neutrophil infiltration and decreased expansion and inflammatory activation of recruited F4/80lo‐MHC‐IIhi peritoneal macrophages. Together, these results uncover a previously unrecognized function for LXRα‐dependent transcriptional cis‐activation of secondary inflammatory gene expression in macrophages and the host response to microbial ligands.


Introduction
The inflammatory response is a defense mechanism of the innate immune system against infections and other injuries.Inflammation is rapidly initiated aiming to isolate and eliminate the damaging stimuli, leading to tissue repair and healing to restore homeostasis. [1]Thus, an effective inflammatory response must be robust enough, but temporarily restricted until the injury has been eliminated.Although inflammation is a crucial protective reaction, uncontrolled chronic inflammation can cause or aggravate several life-threatening diseases, including cancer, diabetes or cardiovascular diseases. [2,3]acrophages are phagocytic cells that are present in all tissues and orchestrate decisive steps at all stages of inflammation.In response to infection, macrophages detect pathogen molecular structures by innate sensors, such as Toll-like receptors (TLR) that recognize lipopeptides, singlestranded DNA or double-stranded RNA. [4]ngagement of TLRs and the adapter proteins Myeloid differentiation primary response 88 (MyD88) and Toll/Interleukin-1 receptor domain-containing adapter protein inducing Interferon beta (TRIF) leads to activation of NF-B, AP-1, and IRF transcription factors (so-called ´Signal-Dependent Transcription Factors´, SDTFs), which rapidly promote the expression of many cytokines and soluble factors, such as interleukins and interferons. [5]This primary set of autocrine and paracrine signaling proteins include type I interferons (IFN/), which in turn promote the expression of many secondary-response genes, [5,6] including additional waves of cytokines and genes encoding for enzymes that produce reactive species important for pathogen killing. [5,7,8][11][12] Thus, the transcriptional response of macrophages to microbial components is accomplished by sequential waves of gene induction controlled by different combinations of SDTFs and other transcriptional regulators.
The liver X receptors (LXR, encoded by the gene Nr1h3 and LXR, encoded by the gene Nr1h2) are members of the nuclear receptor superfamily of transcription factors that play crucial roles in sterol homeostasis in mammals, and also regulate inflammation and immunity. [13]LXRs operate as obligate heterodimers with the retinoid X receptors and their endogenous ligands include various intermediates of the cholesterol biosynthetic pathway. [10]Much of the LXR pharmacology and target-gene discovery, however, has been studied with potent, non-steroid synthetic ligands. [14]Mechanistically, LXRs positively regulate gene expression through direct binding to cognate DNA response elements (LXREs) within cis-regulatory regions (i.e., enhancers and promoters) of target genes. [14,15][18] Attenuation of inflammation is observed when synthetic LXR ligands are administered before the injury, as reported in cellular and in vivo models of inflammation. [16,17,19]Restraining inflammation could be beneficial in certain settings, but may also weaken the host defense against infections, as shown by synthetic LXR ligands during pulmonary infection. [20,21]Moreover, alleviation of inflammation exerted by synthetic LXR ligands contrasts with the protection against pathogens demonstrated by endogenous LXR activity in vivo in infection models.LXRdeficient mice present defective innate immunity against Listeria monocytogenes [22] or Mycobacterium tuberculosis [23] and an inade-quate response to cecal ligation and puncture model of polymicrobial sepsis, [24] suggesting that endogenous LXR activity, instead of repressing, potentiates innate immune responses.
LXR-dependent immune protection against bacterial infection has been shown to be mediated mainly by LXR. [22,23]However, the molecular connections between innate immune pathways and endogenous LXR activity in macrophages have not been explored in depth.The objective of this study was to investigate the transcriptional regulation, differential DNA binding and in vivo activity of LXR in response to a prototypical microbial component, the TLR4 agonist LPS.Using primary macrophages with genetic inactivation of key inflammatory signaling components, transcriptional profiling and ChIP-seq studies, we demonstrate that TLR4 signaling induced a secondary inflammatory response that involves LXR transcriptional induction, which in turn rewires its genomic binding landscape to cis-regulatory regions of inflammatory genes, whereby it promotes cytokine and chemokine gene expression.These data uncovered a novel TLR4-LXR axis that sustains macrophage inflammatory gene expression and in vivo immune-cell recruitment during inflammatory responses to microbial ligands.

Microbial Components Induce LXR𝜶 Transcription during Secondary Inflammatory Responses
Previous work from our group and others demonstrated that macrophage LXRs participate in the regulation of inflammation and immunity, [16][17][18] but the impact of microbial ligands and their signaling pathways on endogenous LXR transcriptional activity has not been explored in depth.Also, in a former study using ectopic expression of LXR or LXR in an LXR-deficient background macrophage model, we demonstrated non-overlapping transcriptional actions of each receptor beyond fatty acid and sterol metabolism. [25]Since LXR plays a singular role in the protection against infections, [22,23] we focused our interest on the regulation of Nr1h3 gene transcription (for clarity with the nomenclature, we will use Lxr for the gene/mRNA and LXR for the protein) and LXR protein expression in macrophages activated by microbial ligands.We chose bone marrow-derived macrophages (BMDM) differentiated with MCSF as cell model. [25]Although earlier reports showed increased Lxr mRNA expression in LPS-activated myeloid cells, [26][27][28][29] the molecular mechanisms underlying this induction and the identification of LXR downstream targets in the context of inflammation, have not been thoroughly investigated.To establish a time frame in which Lxr mRNA was induced by TLR agonists in BMDM, compared to other prototypical primary and secondary-responsive genes, we investigated the expression of Tnf, Cxcl10, Nos2 and Lxr genes from public datasets. [30]Activation of TLR2, TLR3, TLR4 and TLR7/8 increased the expression of early and late inflammatory genes with different timing, as expected (Figure 1A).While Tnf expression acutely raised during the first hour post-challenge, TLR-dependent expression of Lxr augmented several hours later, similar to other secondary-responsive genes, such as Nos2 (Figure 1A; Figure S1A, Supporting Information).Our experiments reproducibly showed maximal induction of Lxr mRNA at 18-24 h post LPS, whereas Lxr expression did not change substantially (Figure 1B).
LXR protein levels gradually increased in LPS-stimulated macrophages, similar to NOS-2 (Figure 1C).LPS induction of Lxr mRNA showed LPS dose-dependency and changes in Lxr mRNA or nascent hnRNA were abolished in TLR4 -/macrophages (Figure S1B and C, Supporting Information).The transcription inhibitor Actinomycin D declined both Lxr mRNA and nascent hnRNA after the LPS challenge, consistent with the notion that TLR4 signaling directly impacts Lxr primary transcription and not mRNA stability (Figure S1D, Supporting Information).Moreover, experiments employing protein synthesis inhibitor cycloheximide for different times decreased the LXR protein levels that were induced by LPS to a similar ratio as control cells (Figure S1E, Supporting Information).This implies that the amount of accumulated LXR protein in response to prolonged periods of TLR4 stimulation was not due to protein stabilization.Surprisingly, although Lxr mRNA and protein levels potently augmented in response to LPS, the expression of classic LXR target genes did not parallel this temporal pattern.Indeed, the expression of many LXR targets, with the exception of Cd38, increased at early times in response to LPS, and returned to control levels or decreased at the time LXR protein was maximal (Figure 1C,D).As previously reported, [31] we confirmed that the expression of the cholesterol 25 hydroxylase (Ch25h, which catalyzes the conversion of cholesterol into 25-Hydroxycholesterol, 25-HC) was also potently induced by LPS, and would be a potential source of the oxysterol ligand 25-HC [32] during inflammation (see below, also connected to Figure 4; Figure S3, Supporting Information).Together, these experiments revealed that LXR expression, but not LXR, was induced during extended phases of the inflammatory response in macrophages but its endogenous activity was not linked to positive regulation of established LXR target genes.

Intracellular Determinants Responsible for LXR𝜶 Expression in Inflammatory Macrophages
The observation that Lxr expression was induced by several TLR agonists led us to investigate common intracellular pathways downstream of TLRs that might be responsible for this activation.First, we examined the signaling downstream MyD88, a common adaptor protein crucial for most TLR-dependent signals.Analysis of gene expression using MyD88-/-macrophages revealed that, while Il1b expression was blunted, induction of Lxr was similar in WT and MyD88-/-macrophages (Figure 2A).To refine the search for intracellular transduction signals that trigger Lxr expression downstream of TLRs, we analyzed the effect of a battery of validated inhibitors that target key signaling molecules.Blocking the activation of IKK or TAK1 decreased Lxr expression in response to LPS (Figure 2B), indicating that activators of the NF-B pathway participated in this regulation.Secondary inflamma-tory responses require autocrine activation by cytokines, such as TNF, which in turn directly activates the NF-B pathway. [33]We employed TNF-deficient macrophages to explore this possibility.The analysis revealed similar expression of Lxr in both WT and Tnf-/-macrophages in response to LPS or Poly I:C, indicating that endogenous TNF was not required for the late induction of LXR (Figure 2C).
To explore the potential SDTFs that could be participating in the control of LXR expression in response to inflammatory signaling, we compared the recruitment of key SDTFs to promoter regions of early (Infb1) or late (Lxr) response genes.Analysis of public ChIP-seq datasets [11,12,34,35] revealed that Infb1 promoter, but not Lxr, was pre-marked with potent IRF-8 binding.In addition, IRF-1, IRF-3 and p65 NF-B were recruited earlier to the Infb1 promoter in response to TLR4 signaling, whereas binding of p65 NF-B, but not IRFs, was observed later in response to LPS within the Lxr enhancer region (Figure 2D).When comparing the genomic recruitment of SDTFs in response to INF stimulation, to mimic autocrine secondary response to type I IFNs, we did not observe STAT1 binding to the Ifnb1 promoter, but prominent recruitment of p65 NF-B and STAT1 at the Lxr enhancer region.In addition, canonical RELA NF-B and STAT binding motifs were found at the Lxr regulatory region (Figure 2D, right).These results suggest that NF-B and STATs are important for the secondary induction of Lxr expression by TLR signaling.

Inducible LXR𝜶 Expression Requires TRIF/TBK-1/IRF-3 Activation and Type-I Interferons
Given the possible involvement of type I IFNs in the temporal induction of Lxr expression by LPS, we studied its regulation by the TANK-binding kinase 1 (TBK-1) and Interferon regulatory factor 3 (IRF-3) pathway, which is crucial for autocrine and paracrine IFN signaling. [8,36]Lxr mRNA expression was impaired in Irf3-/-BMDM compared to WT cells in response to LPS (Figure 3A, left panel).In addition, macrophages cultured with TBK1 inhibitor MRT67307 [36,37] partially blocked Lxr induction in WT cells and had little effect in Irf3-/-cells.The analysis of LXR expression revealed that, although LPS-induced LXR levels were mostly dependent on IRF-3 expression, there was some remaining LXR expression in Irf3-/-macrophages, suggesting that additional TFs were involved in the regulation of LXR protein expression (Figure 3A, right panel).To directly address the role of type I IFNs in LXR induction, we analyzed LXR expression in response to LPS by real-time qPCR and western blot in macrophages deficient in type I IFN receptor, IFNAR-1 (Figure 3B).This analysis revealed that Lxr mRNA (Figure 3B, left panel) and protein levels (Figure 3B, right panel) were severely impaired in Ifnar1-/-macrophages compared to WT, confirming that secondary signaling driven by type I IFNs was crucial for triggering inducible LXR expression.Moreover, using Irf1-/-, Irf3-/-and Stat1-/-macrophages, we corroborated Figure 1.A) Time-course of mRNA expression of the indicated genes in response to TLR agonists: LPS (TLR4), Poly I:C (TLR3), PAM3CSK4 (TLR2) and R848 (TLR7 and TLR8); normalized values of RNA expression were obtained from database ArrayExpress E-TABM-310. [30]B) Relative mRNA expression of Lxr and Lxr in BMDMs cultured 24 h with LPS (100 ng ml −1 ).C) Protein levels of LXR, ABCA1 and NOS2 in WT BMDMs cultured with LPS (100 ng ml −1 ) or PBS for different times (0 -48 h).Band intensities quantified by densitometry are displayed below each blot image.D) Relative mRNA expression levels (0 -48 h.) for indicated genes in WT BMDMs cultured with LPS (100 ng ml −1 ).Expression data in B-D) are represented as mean ± SD from n = 3 experiments.Significant differences between mean values with the control condition were indicated (* p < 0.05 and ** p < 0.01).
that inflammatory SDTFs that are activated downstream of type I IFNs were decisive for Lxr mRNA expression (Figure 3C).
Because the MyD88-independent arm of TLR3/4 signaling requires the adaptor protein TRIF [38,39] (also known as TICAM-1), for TBK-1/IRF-3/IFN activation, we analyzed Lxr expression in WT and Trif-/-macrophages.In response to LPS, expression of Lxr was diminished in Trif-/-cells (Figure 3D).Importantly, challenging cells with IFN alone was able to partially rescue Lxr expression in Trif-/-macrophages to similar levels as WT cells, indicating that IFN signaling is important for Lxr expression during secondary inflammatory responses.Collectively, these findings provide evidence that IFN- via TLRs/TRIF/TBK1/IRF3 signaling is crucial for the inducible expression of LXR in macrophages as a secondary-responsive gene to microbial-ligand challenging.

Global Profiling Identifies Specific Subsets of LXR𝜶-Dependent Genes Following TLR4 Activation
To reveal the full spectrum of endogenous LXR activity and its downstream signaling, we performed gene expression profiling in WT and Lxr-/-macrophages cultured 24 h with LPS.Principal component analysis (PCA) of gene expression data showed that replicate profiles of LPS-stimulated Lxr-/-macrophages differed considerably from their WT counterparts (Figure 4A, left panel).Volcano-plot and heat-map analysis revealed clusters of genes differentially regulated (Fold Change [FC] ≥ 2 and P-value < 0.05) in Lxr-/-macrophages in response to LPS.Clustering of differentially expressed genes (FC>2 across genotypes) identified two principal groups of genes (clusters I, II) (Figure 4A right panel and 4B).Cluster I comprised genes that were defectively induced by LPS in Lxr-/-macrophages, whereas cluster II is composed by genes that did not show similar repression in Lxr-/-after LPS treatment, compared to WT macrophages.Gene ontology and pathway analysis of cluster-I revealed enrichment of inflammatory processes such as NF-B signaling pathway, response to interferon and acute phase response (Figure 4C).Indeed, genes that showed the greatest differences in Cluster I corresponded to hallmark proinflammatory cytokines (Tnf, Il6, Il1b and Il1a), and chemokines (Ccl2, Ccl7, Cxcl2 and Cxcl11) (Figure 4B, right panel) that were more expressed in WT compared to Lxr-/macrophages in response to LPS.In contrast, cluster II contained transcripts more expressed in Lxr-/-macrophages and showed enrichment in pathways related to cell-cycle, cellular response to DNA damage and response to wounding (Figure 4B,C).Moreover, we compared the mRNA expression of a panel of selected cytokines, chemokines or anti-microbial genes in WT, Lxr-/-and Lxr-/-BMDM in response to increasing times of LPS stimulation (Figure S2, Supporting Information).The analysis of qPCR data showed that expression of Il1a, Il1b, Il6, Inhba, Marco and Tnf was decreased in Lxr-/-macrophages at prolonged, but not at short times after LPS challenge.Expression of other inflammatory genes, such as Nos2 or Mx1 did not significantly depend on Lxr or Lxr expression after LPS treatment (Figure S2, Supporting Information).
The observation that several inflammatory genes required Lxr expression for their full induction at later, but not early times of LPS stimulation was intriguing.In addition, our previous data presented in Figure 1d showed that early LPS stimulation induced the expression of targets involved in cholesterol or fatty acid metabolism, yet uncoupled to the temporal LXR maximal induction.These facts questioned if perhaps LPS regulation of established LXR targets required both Lxr and Lxr for their expression and whether the oxysterol 25-HC (produced by the prominent induction of Ch25h, Figure 1D and reported before [31] ) would serve as endogenous LXR ligand [40] for the early and late transcriptional actions of LPS signaling.Analyzing independent data from available expression profiling of LPS-stimulated BMDM, [41] obtained from either WT, Lxr,-/or Ch25h-/-mice, showed that expression of most LXR targets was higher in Ch25h-/-in response to LPS (with the exception of Abca1 and Abcg1), but required Lxr and LXR for their full expression (Figure S3, Supporting Information).Interestingly, many of the pro-inflammatory genes identified from Cluster I of our profiling (Figure 4B, which were improperly induced by LPS in Lxr-/-macrophages) were also downregulated in Lxr,-/-, thus reinforcing our conclusions (Figure S3, Supporting Information).In addition, expression of some of these proinflammatory genes, including Inhba, Il6, Ccl2 and Ccl7 was partially downregulated in Ch25h-/-, suggesting that 25-HC might be involved as endogenous ligand targeting LXR for their transcriptional activation.Expression of Il1a, Il1b or Cxcl2, however, was upregulated in Ch25h-/-, indicating that other ligand/s besides 25-HC might be participating as LXR ligands for their regulation.Overall, these results suggested, surprisingly, that the inducible transcription of endogenous LXR at later stages of macrophage responses was not playing an anti-inflammatory role, but rather was important for the expression of a battery of secondary-responsive pro-inflammatory genes.

Deciphering the LXR𝜶 Genomic Binding Landscape in Macrophages in Response to LPS
Previous studies reported that TLR ligands induce partial reprogramming of macrophage SDTFs at predefined genomic cisregulatory locations. [42]To study whether LXR was potentiating secondary inflammation through direct binding to genomic regulatory regions of target genes, we optimized an LXR ChIPseq assay using an LXR-dual polyclonal antibody [43] (Figure S4, Supporting Information) and Lxr-/-macrophages that express  LXR and showed differential regulation of inflammatory gene expression when compared to Lxr-/-cells at 24 h post LPS challenge (Figure S2, Supporting Information).We searched for de novo LXR binding sites induced by LPS compared to control, using Lxr,-/-double deficient macrophages as negative control.Bioinformatic analysis revealed a dynamic occupation of LXR binding sites (1960 genomic regions) in response to late LPS signaling (Figure 5A).Analysis revealed that secondary response to LPS induced robust LXR binding at sites in which LXR was very little present or not present in unstimulated cells, suggesting that a prolonged inflammatory response promoted a novel LXR binding landscape distinct from the vicinity of classic, sterol metabolic LXR target genes (Figure 5A; Figure S5A, Supporting Information).Indeed, LXR binding at classic target-gene loci did not change significantly after LPS stimulation (Figure S5A, Supporting Information, left panel) and suggested that newly synthesized LXR might act as an inflammatory SDTF in combination with other transcription factors.
To gain insight into additional transcription factors that might bind to those regulatory regions in combination with LXR, we studied the sequence patterns enriched within LXR-binding sites by motif analysis (Figure 5B).This revealed that LXR preferentially binds regions enriched for the canonical LXR binding site (LXRE) or modified LXR sites containing a half-site nuclear receptor binding motif (AGGTCA), along with binding sites for the master macrophage LDTF PU.1 and inflammatory SDTFs including ATF/AP1 family members, NF-B and IRFs (Figure 5B).Analysis of gene ontology and biological pathways of LXR genomic-binding sites revealed enrichment of the inflammatory response and myeloid leukocyte migration (Figure 5C).We reasoned that LPS-inducible, chromatin-bound LXR, might help promoting late inflammatory gene expression through collaboration with other SDTFs.We focused on NF-B as this TF appeared enriched in both, the GO biological pathways of Cluster I of genes defectively induced by LPS in Lxr-/-macrophages (Figure 4C), and the motif analysis of adjacent sequences to those were newly synthesized LXR was deposited (Figure 5B).To test the possible influence of LXR on NF-B recruitment at cisregulatory regions of inflammatory genes, we performed ChIPseq analysis of the p65 component of NF-B in WT and Lxr-/-macrophages at early (3 h) and late (24 h) stimulation with LPS (Figure 5A,D,E; Figures S5 and S6, Supporting Information).Long treatment for 24 h induced a large number of accessible chromatin regions with p65 binding (total 7187 peaks).When we looked at the p65 binding peaks that were aligned with the LXRsensitive regions, we observed that an important proportion of p65 binding had decreased in Lxr-/-macrophages as compared with WT macrophages at late LPS times (Figure 5E; 765 out of 1960).This indicates that ≈40% of the regions co-occupied by LXR and p65 in WT cells in response to LPS experienced an important reduction of NF-B binding in Lxr-/-macrophages (Figure 5A,D).Examples of genomic loci which showed inducible LXR binding in response to LPS that displayed a reduction of p65 binding in Lxr-/-macrophages include enhancer regions important for Il1a, Il6 or Ccl2/Ccl7 expression.In contrast, those same locations presented a similar p65 binding at at 3 h post-LPS stimulation in WT and Lxr-/-macrophages (Figure S6, Supporting Information).
We also employed ChIP-seq to study the H3K27Ac acetylation signal, which has been directly associated with active enhancers [44,45], using WT and Lxr-/-macrophages.As shown in Figure 6A, 660 enhancer regions showed defective H3K27Ac up-regulation in Lxr-/-macrophages in response to LPS.Genomic regions in this cluster included regulatory vicinities of genes coding for CCLs, CCRs and CXCLs chemokines, which are associated with functions in defense response, cytokine response and neutrophil recruitment.Given the important crosstalk of LXR and p65 NF-B TFs at genomic regions that were reprogrammed after long LPS stimulation times, we aligned discrete enhancer regions that either gained or lost H3K27ac marks with those LXR and p65 NF-B peaks (Figure 6B, scatter plot).Upregulated H3K27ac regions induced by LPS (marked in red in Figure 6B) contained more LXR (31 vs. 18) and p65 (132 vs. 24) binding sites than regions that exhibited decreased H3K27ac marks (marked in blue in Figure 6B), consistent with the hypothesis that the co-occurrence of LXR and NF-B was important within genomic regions associated with transcriptional activation during inflammation.In addition, examples of discrete enhancer regions in the vicinity of the Ccl2, Il1r1 and Abcg1 loci corroborated the proposed mechanism that operates within inflammatory or non-inflammatory genes (Figure 6C; Figure S5B, Supporting Information).A group of enhancers that control the expression of cytokines and chemokines showed greater H3K27Ac signal in response to LPS in WT BMDM, which was diminished in Lxr-/-macrophages, possibly due to reduced recruitment of p65 NF-B and other transcriptional coregulators, resulting in decreased histone acetylation and inadequate gene activation (Figure 6C, right panel and).On the other hand, classic LXR targets, such as Mylip or Abcg1, showed unaltered LXR binding in response to LPS, low or no recruitment of p65 NF-B and minimal changes in H3K27Ac (Figure 6C; left panel and Figure S5A, Supporting Information left panel).Together, ChIP-seq and transcriptional profiling data showed that LPS-inducible LXR preferentially binds to canonical LXRE sequences that are predetermined by macrophage LDTFs which, in combination with p65 NF-B recruitment and other SDTFs inflammatory transcription factors, promoted secondary inflammatory gene expression.

Endogenous LXR𝜶 Facilitates Immune-Cell Recruitment at Inflammation Sites In Vivo
The in vitro studies presented above indicated that LXR participates in the maintenance of macrophage secondary inflammatory response induced by TLR stimulation.Inflammatory mediators, such as cytokines and chemokines are important for the recruitment of immune cells to sites of infection and injury. [2]o investigate the role of endogenous LXR-dependent pathways in modulating inflammation in vivo, we used a validated model of murine peritoneal inflammation using three different challenges; [46,47] LPS, zymosan or thioglycollate were injected into cohorts of WT and LXR deficient mice.To concentrate our analysis on the role of macrophages and to avoid the contribution LXR expressed in other cells, such as hepatocytes in the liver, we employed a previously characterized C57Bl6 mouse model with LXR deficiency in hematopoietic cells (Lxr fl/fl -iVav-Cre + ) that showed potent recombination in macrophages in vivo. [48]As a readout of the inflammation status, we used the accumulation of neutrophils in the peritoneal cavity at 24 h post-injury. [49]As expected, a prominent accumulation of neutrophils (peritoneal cells with surface expression of CD11b + /Ly6G + ) into the peritoneal cavity of WT mice emerged 24 h post-challenge in response to all three peritonitis insults (Figure 7A).Strikingly, while thioglycollate and zymosan treatments did not reveal significant differences in neutrophil frequency between Lxr fl/fl -iVav-Cre − (WT) and Lxr fl/fl -iVav-Cre + (Figure 7A,B), a marked reduction in neutrophils was observed in peritoneal exudates from Lxr fl/fl -iVav-Cre + mice that were challenged specifically with LPS.These results suggest that endogenous macrophage LXR plays an unexpected role in controlling the infiltration of immune cells that are recruited to sites of inflammation in response to specific microbial stimuli, such as LPS.
To explore in depth a possible differential macrophage activation in vivo in the peritoneum that might explain the distinct responses to peritonitis models in the absence of Lxr, we analyzed the frequency and the transcriptional phenotypes of peritoneal macrophages.Under steady-state conditions, the majority of resident peritoneal macrophages (also called large peritoneal macrophages) express high levels of CD11b and F4/80, but low MHC-II (designated here as F4/80 hi /MHC-II lo ). [47]The second subset, expresses low levels of F4/80 but expresses high levels of MHC-II, designated here F4/80 lo /MHC-II hi (also referred to as small peritoneal macrophages). [46,47]First, to examine the role of myeloid LXR in the differentiation and maintenance of subsets of peritoneal macrophages, we performed flow cytometry analysis of resting peritoneal macrophages from Lxr fl/fl -iVav-Cre − (WT) and Lxr fl/fl -iVav-Cre + (gating strategy, Figure S7, Supporting Information).Mice deficient in LXR in hematopoietic cells do not show major differences in the frequency of peritoneal F4/80 hi /MHC-II lo or F4/80 lo /MHC-II hi macrophage subsets (Figure S7, Supporting Information).In response to inflammatory peritonitis models, the proportion of F4/80 hi /MHC-II lo population declines significantly after the challenges, consistent with a previously described reaction known as the "macrophage disappearance reaction". [47,50]The reduction in F4/80 hi /MHC-II lo population in response to thioglycollate and zymosan was similar in Lxr fl/fl -iVav-Cre − (WT) and Lxr fl/fl -iVav-Cre + mice (Figure 8A,B).Remarkably, however, we observed differences in the profile of peritoneal macrophages when mice were challenged with LPS.The proportion of F4/80 lo /MHC-II hi macrophage subset increased with LPS in Lxr fl/fl -iVav-Cre − (WT) but not in Lxr fl/fl -iVav-Cre + mice (Figure 8A,B).Therefore, in the absence of LXR, inflammatory F4/80 lo /MHC-II hi macrophages did not expand in the peritoneal cavity to the same extent as in WT mice in response to 24 h of LPS, which may have an impact in the inflammatory mediators expressed by macrophages and the differential recruitment of neutrophils observed in the absence of LXR.This result is consistent with the hypothesis that Lxr-/-macrophages, in an in vivo context, do not acquire the appropriate inflammatory phenotype in response to LPS compared to WT cells.
We further investigated whether the deficit in frequency observed in the inflammatory F4/80 lo /MHC-II hi macrophage subset in the absence of LXR would phenocopy some of the characteristics that were observed in vitro, and might present a differential expression of genes involved in inflammation and chemotaxis.We then purified F4/80 lo /MHC-II hi macrophages from Lxr fl/fl -iVav-Cre − (WT) and Lxr fl/fl -iVav-Cre + mice in response to LPS challenge by FACS sorting, and analyze their gene expression by RNA-seq (Figure 8C).Analysis of differentially expressed genes identified Tnf, Il1a, Il1b, Ccl2, Ccl5 and Cxcl10, Ptges and Ptges2 whose expression was higher in LPS-activated Lxr fl/fl -iVav-Cre − WT macrophages compared to Lxr fl/fl -iVav-Cre + cells.These inflammatory genes encode for chemokines, cytokines or cyclooxygenases and prostaglandin synthases which are known to mediate the infiltration of inflammatory cells into sites of injury.These results support the conclusions obtained in vitro with BMDM and emphasize the importance of LXR expression in macrophages to acquire a competent expansion and phenotypic activation in vivo in response to bacterial LPS to support the maintenance of secondary inflammatory responses.Together, the results obtained in mice with the peritonitis models identified LXR as a principal factor in macrophages that sense and amplify the inflammatory response triggered by microbial ligands in vivo.

Discussion
Macrophages rapidly detect infections and trigger sequential waves of anti-microbial responses that affect the expression of hundreds of genes. [10,51]To facilitate induction of gene expression, chromatin organization must be transformed from repressed basal conditions to allow the binding of SDTFs that drive inflammation-dependent transcription. [52]While the role of preexisting transcription factors such as NF-B, AP-1 or IRF-3 has been extensively studied in macrophages during the initial activation phase, [6,42,52,53] less is known about factors that are transcriptionally induced at later phases of inflammatory activation.The studies presented here unveil a previously unrecognized, cell-autonomous role for LXR in macrophage inflammatory activation.Induction of LXR transcription is an important component of macrophage secondary responses that amplify initial inflammation.NF-B, IRF-3 and type I IFNs cooperate in LXR transcriptional induction, which in turn, facilitates the extension of inflammatory and anti-microbial responses.We show that endogenous LXR activity sustains the expression of cytokines and chemokines during inflammation and enables immune-cell recruitment and activation in response to endotoxin in vivo.Our studies also demonstrate that endogenous LXR positively regulates secondary inflammatory gene expression through direct Figure 5. A) Heatmap representation of normalized tag densities obtained through LXR ChIP-seq around 2 Kb of de novo LXR binding locations in Lxr-/-BMDMs cultured with or without LPS for 24 h (density map represented in green).As negative control, ChIP-seq was performed in Lxr,-/-BMDM (left panels, in red).Parallel heatmap representation of p65 NF-B ChIP-seq in WT and Lxr-/-BMDMs cultured with or without LPS for 24 h (right panels, in blue).B) Sequence motif analysis associated with de novo LXR peaks in LXR-/-BMDMs cultured with or without LPS for 24 h.C) Gene Ontology (GO) analysis associated with de novo LXR peaks.D) Distribution of ChIP-seq tags surrounding the LXR peak centers.E) Venn-diagram representation of total number of LXR and p65 NF-B peaks obtained by ChIP-seq analysis of BMDM challenged with LPS for 24 h.binding to cis-regulatory regions of target genes.This facilitates the recruitment of the p65 component of NF-B and possibly other inflammatory SDTFs, that cooperatively promote histone H3K27 acetylation and transcriptional activation.The ability of endogenous LXR to positively modulate inflammatory gene expression at later stages of anti-microbial responses provides a plausible mechanism by which endogenous LXR contributes to innate immune functions.
The role of LXRs in inflammation and host immunity has been the subject of intense investigation since the original studies by some of the authors (AC, PT). [16,17,54][57] In addition, LXR acprimarily through LXR, supports innate immunity in murine models of infection, [22][23][24] but pretreatment with synthetic LXR ligands may not be beneficial for host immunity as they dampen anti-microbial inflammatory cascades. [20]Thus, the anti-inflammatory activity of LXR ligands can be dissociated from the innate immune functions driven by endogenous LXR signaling.Our present studies shed light on the function of LXR in macrophages exposed to microbial ligands in the absence of synthetic LXR agonist supplementation.How can we reconcile the literature and our past studies presenting LXR agonists as anti-inflammatory agents with our current work that shows endogenous LXR expression sustaining inflammation?First, the anti-inflammatory actions of LXR ligands have been welldocumented when agonists are administered hours/days before the insults as preventive therapy. [16,17,58]Second, studies demonstrated that repression of inflammation by LXR ligands is dependent on transcriptional induction of ABCA1, [50,59,60] which in turn is expressed at membrane lipid microdomains, uncoupling TLR signaling and downregulating NF-B and MAPK activation. [58,61]ndeed, macrophage-specific ABCA1 deficient-mice display increased inflammation and enhanced ability to fight infection. [59][64] Thus, pharmacological induction of LXR targets by pre-treatment with synthetic agonists limits subsequent inflammation.
Prior studies revealed the importance of LXR-dependent gene expression and LXR synthetic ligands as anti-inflammatory agents. [13]However, during macrophage physiological activation, endogenous LXR activity can be regulated by changes in receptor expression and the availability of sterol-derived ligands, [14,15] processes that are both differentially regulated during inflammation.][72][73] A notable exception is the Ch25H, whose expression in macrophages is robustly induced (Figure 1D) in a type I interferon-dependent manner. [68]nduction of 25HC production exerts potent anti-viral actions and appears to stimulate immune activation. [67,68,74]n addition, the accumulation of sterol intermediates during macrophage inflammation or foam-cell formation has been reported to exert different responses, including anti-inflammatory and anti-microbial, which have shown different degrees of LXR-dependency. [65,67,75]Our study adds new knowledge about LXR expression, its epigenomic regulation and function during inflammation.Our analysis of RNA-seq data from Ch25h-/-macrophages showed that some of the inflammatory genes identified as LXR targets in response to LPS were also dependent on CH25H expression.Thus, changes in levels of various intermediates in the cholesterol biosynthesis or postcholesterol oxysterols may have LXR-dependent and independent outcomes under different temporal dynamics of macrophage activation.Alternatively, we recently described an LXR transcriptional mode of action that is pharmacologically insensitive to agonist modulation, [25] suggesting that LXR could additionally function in an LXR ligand-independent manner under different macrophage pathophysiological settings.In this work, our gene expression analysis adds that, while LXR transcription is induced to sustain TLR-dependent macrophage activation, ABCA1 mRNA and protein, and several other LXR targets did not follow such rate of expression.Considering previous literature and new evidence presented here, we propose that TLR-dependent temporal stimulation of LXR expression, along with antagonism of classic LXR targets (that limit their anti-inflammatory activity), favors a greater magnitude of inflammation during prolonged macrophage response to microbial pathogens with a direct implication of LXR.
Recent studies have documented the genomic landscape of both LXR/LXR in thioglycollate-elicited peritoneal macrophages or Kupffer cells using ChIP-seq and dual LXR, antibodies, [76][77][78] but the individual LXR and LXR genomic binding profiles under different macrophage conditions are still poorly defined.While many LXR transcriptional functions are redundantly performed by LXR or LXR in cultured macrophages in response to synthetic agonists, a unique role of LXR in the differentiation of liver and spleen resident macrophages has emerged. [48,77,78]LXR is highly expressed in several tissue macrophage subtypes and drives the expression of myeloid genes in response to in vivo-derived ligands.This suggests that signals and pathways that induce LXR expression and promote  the production of sterol derivatives in local microenvironments may have distinctive pathophysiological implications. [69]t will be important for future investigations to study LXR transcriptional and epigenomic properties in tissue-resident macrophages under homeostatic or pathological scenarios.Our present contribution, using BMDM that express comparable levels of both LXRs under basal conditions, [16] describes a specific mechanism by which TLR-induced LXR directly binds to regulatory regions of inflammatory genes and stimulates their expression.In resting conditions, LXR genomic binding sites are mainly found within the vicinity of genes involved in sterol and fatty acid metabolism.In response to TLR activation, NF-B, IRF-3 and STAT-1 coordinately facilitate new LXR transcription.Intriguingly, while the total LXR protein increased robustly after LPS stimulation, binding of LXR did not change considerably in LXR classic target-loci but instead appears notably recruited to regulatory regions of inflammatory genes.Our motif enrichment analysis indicates that newly-synthesized LXR appears within predetermined, or ´poised´, IRF-8/PU.1+genomic loci.The observation that transcripts induced by LPS in WT macrophages through these collaborative SDTFs along with LXR binding, but whose expression was attenuated in LXR-/-macrophages, strongly suggests a direct role for LXR in their positive regulation during inflammation.Recruitment of LXR to inflammatory genes facilitates the maintenance of p65 component of NF-B at prolonged times after LPS stimulation, presumably in cooperation with other inflammatory SDTFs including IRF-1 and IRF-3.Consistent with these results, regions with higher H3K27ac in WT macrophages, associated with active enhancers, were repressed in many inflammatory genes and presented less binding of p65 NF-B in LPS-stimulated Lxr-/macrophages, suggesting that Lxr expression favors the permanency of NF-B and histone acetyl-transferase complexes within those enhancers during secondary inflammatory responses.The LXR-regulated transcriptional activation mechanisms uncovered in cultured macrophages were also validated in vivo with peritonitis models.Studies on the transcriptional components that are involved in macrophage inflammatory response established a coordinated collaboration between different SDTFs that bind to genomic cis-regulatory regions. [5,6,42]Our work on LXR extends the repertoire of inflammatory SDTFs that collaborate in macrophage secondary inflammatory response and highlights the importance of LXR signaling in host immunity against pathogens.In summary, our findings position LXR activity at center stage of transcriptional regulation of inflammation and provide a plausible mechanism by which endogenous LXR contributes to anti-microbial responses.
RNA and Protein Analysis: Whole-cell lysis was performed with radioimmunoprecipitation assay buffer [25] (RIPA; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors, from Roche).Protein extracts were subjected to SDS-PAGE and transferred to nitrocellulose or polyvinylidene difluoride (PVDF) membranes (Bio-Rad).Primary antibodies were described above.Reactive bands were detected by Clarity Western ECL substrate (Bio-Rad).For RNA studies, total RNA was extracted from cells using TRIZOL Reagent (Invitrogen) following manufacturer guidelines.RNA was dissolved in DEPC-H2O, and 1 μg of RNA was used for iScript cDNA synthesis (Bio-Rad).For real-time quantitative PCR, cDNA was used along with 2X PCR MasterMix (Diagenode) specific primer mix.Primer sequences were displayed in Table S1 (Supporting Information).Fluorescence emission in real-time and analysis was performed with a CFX thermal cycler (Bio-Rad).The relative levels of RNA were measured following the ΔΔCT method and individual expression data were normalized to 36B4 expression.
Chromatin Immunoprecipitation (ChIP) Assay: Cell fixation and crosslinking were performed as follows: First, sets of 5-7 × 10 6 macrophages Figure 8. A) Flow cytometry analysis of peritoneal exudates showing subpopulations of peritoneal macrophages in Lxr fl/fl -iVav-Cre − (WT) and Lxr fl/fl -iVav-Cre + mice, identified as shown in Supporting Figure 7, in control mice and in response to peritonitis stimuli: LPS (4 mg kg −1 per mouse), thioglycollate (2 ml of 3% solution per mouse) and zymosan (1 mg per mouse).B) Quantification of flow cytometry data showing the percentage of each macrophage subpopulation among total peritoneal cells.Flow cytometry data were represented as mean ± SD from one or two experiments with n = 4-5 per group.Significant differences between mean values are denoted with asterisks (** p < 0.01).C) mRNA data obtained from FACS-sorted F4/80 lo /MHC-II hi inflammatory macrophages purified from mice that were challenged with LPS for 24 h.Purified cells were obtained from pools of n = 4 mice per genotype.Expression values obtained from bioinformatic analysis of RNA-seq data were represented as Transcripts Per Million reads (TPM).

Figure 3 .
Figure 3. A) Left panel.Relative mRNA expression of Lxr in BMDMs from WT or Irf3-/-mice cultured with LPS (100 ng ml −1 for 0 -24 h.) alone or combined with MRT67307 (TBK1 inhibitor, 2 μM), and protein levels (A, right panel) of LXR and GAPDH from cells under the same conditions as in A; relative band intensities were quantified by densitometry.B) Left panel.Relative mRNA expression of Lxr in WT or Ifnar-/-BMDMs cultured with LPS (100 ng ml −1 ) for 24 h, and protein levels (right panel)of LXR, NOS-2 and GAPDH; relative band intensities were quantified by densitometry.C) Relative mRNA expression of Lxr in WT or Ifnar-/-, Stat1-/-, Irf3-/-and Irf1-/-BMDMs cultured with LPS (100 ng ml −1 ) for 24 h.D) Relative mRNA expression of Lxr in WT or Trif-/-BMDMs cultured with LPS (100 ng ml −1 ) or IFN (500 U ml −1 ) alone or combined.All mRNA expression data were represented as mean ± SD from three experiments.Asterisks indicate significance between with WT and knockout cells B-D).Hashes and crosses indicate significant differences in Trif-/-BMDM treated with IFN or LPS+ IFN compared to Trif-/-BMDM treated with LPS alone.Significant differences between means (**, ## or † † p < 0.01).

Figure 4 .
Figure 4. A) Principal Component Analysis (PCA) of detectable mRNAs (left graph) and volcano plot (right graph) of transcriptional profiling data comparing WT and Lxr -/-BMDMs cultured with LPS (100 ng ml −1 ) for 24 h.Differentially expressed genes (p-value < 0.05, FC > 2) are shown in red (Up-regulated) or blue (Down-regulated) in Lxr-/-.Data are from two experiments with n = 2 biological replicates per group, and B) Heatmap plots showing differentially expressed genes among replicates and details of top regulated genes in Cluster I. C) Bar graph of Gene Ontology (GO) enrichment analysis for differentially expressed genes red (Down-regulated) or blue (Up-regulated) in Lxr -/-BMDMs.

Figure 6 .
Figure 6.A) Heatmap representation of normalized tag densities of H3K27ac genomic regions, obtained through ChIP-seq analysis in control versus LPS WT and Lxr-/-BMDM.Each row is z-score normalized tag counts for a peak.Data from n = 3 biological replicates per group.Color codes indicate significant changes (p-adj < 0.05, FC > 2) in H3K27ac with or without LPS.B) Scatterplot of discrete H3K27ac regions in WT versus Lxr-/-BMDM cultured with LPS (cluster regions from A), and their associated LXR (green) or p65 NF-B (purple) binding sites.Data are from n = 3 per group.C) IGV genome browser images for the indicated genomic regions showing LXR, p65 NF-B or H3K27ac peaks in the indicated BMDMs cultured with or without LPS for 24 h.Bar graphs illustrate H3K27ac normalized tag counts for the indicated genomic regions in WT and Lxr-/-BMDMs cultured with LPS for 24 h.Normalized tag counts data were represented as mean ± SD from three experiments.Significant differences between means values were indicated (* p < 0.05 and ** p < 0.01).

Figure 7 .
Figure 7. A) Flow cytometry analysis of peritoneal exudates showing Ly6G + /CD11b + neutrophil accumulation in Lxr fl/fl -iVav-Cre − (WT) and Lxr fl/fl -iVav-Cre + mice injected with different stimuli: LPS (4 mg kg −1 per mouse), thioglycollate (2 ml of 3% solution per mouse) and zymosan (1 mg per mouse).B) Quantification of flow cytometry data showing the percentage of neutrophils among total peritoneal cells.Flow cytometry data were represented as mean ± SD from one or two experiments with n = 4-5 per group.Significant differences between mean values are denoted (** p < 0.01).