Lysosome-Dependent LXR and PPARδ Activation Upon Efferocytosis in Human Macrophages

Efferocytosis is critical for tissue homeostasis, as its deregulation is associated with several autoimmune pathologies. While engulfing apoptotic cells, phagocytes activate transcription factors, such as peroxisome proliferator-activated receptors (PPAR) or liver X receptors (LXR) that orchestrate metabolic, phagocytic, and inflammatory responses towards the ingested material. Coordination of these transcription factors in efferocytotic human macrophages is not fully understood. In this study, we evaluated the transcriptional profile of macrophages following the uptake of apoptotic Jurkat T cells using RNA-seq analysis. Results indicated upregulation of PPAR and LXR pathways but downregulation of sterol regulatory element-binding proteins (SREBP) target genes. Pharmacological inhibition and RNA interference pointed to LXR and PPARδ as relevant transcriptional regulators, while PPARγ did not substantially contribute to gene regulation. Mechanistically, lysosomal digestion and lysosomal acid lipase (LIPA) were required for PPAR and LXR activation, while PPARδ activation also demanded an active lysosomal phospholipase A2 (PLA2G15). Pharmacological interference with LXR signaling attenuated ABCA1-dependent cholesterol efflux from efferocytotic macrophages, but suppression of inflammatory responses following efferocytosis occurred independently of LXR and PPARδ. These data provide mechanistic details on LXR and PPARδ activation in efferocytotic human macrophages.


INTRODUCTION
Macrophage (Mj) engulfment of apoptotic cells (AC), a process known as efferocytosis, promotes resolution of inflammation and tissue repair, while restricting autoreactive immune responses (1). Efferocytosis is a coordinated sequence of events, which starts with recognition of AC and culminates in their phagocytosis, followed by phagolysosomal processing (2). Effective clearance of AC is essential for maintaining tissue homeostasis. More recent studies pointed to its importance in resolution of inflammation, immune tolerance, and cancer development (3). Therefore, a better understanding of efferocytosis is key to comprehend major pathophysiological processes.
Phagolysosomal processing of AC is central to handle ingested material (4,5). It creates an overload with macromolecular species that Mj either use or efflux (2). For example, efferocytotic accumulation of lipids from engulfed cells generates ligands for nuclear receptors, which regulate lipid metabolism in Mj, including liver X receptors (LXRa and LXRb) and peroxisome proliferator-activated receptors (PPARa, d or g) (6). LXRs, PPARg and PPARd are wellcharacterized transcriptional regulators that coordinate the clearance of AC as well as anti-inflammatory responses of Mj (7)(8)(9).
Most of the previous work concerning the roles of LXRs and PPARs in efferocytosis employed animal models, often using mice with a Mj-specific deficiency of individual transcription factors (7,8). LXRa/b-and PPARd-deficient Mj reduce expression of the efferocytotic receptor Mer to about 25 to 40%, which largely attenuates the uptake of AC (7,8). Thus, gene expression changes due to a constitutive deficiency of nuclear receptors substantially alter efferocytotic capabilities of Mj. Therefore, model systems using a gene knockout strategy are only of limited predictive value when addressing the relevance of acute activation of LXRs and PPARs during efferocytosis. Along these lines, mechanistic work on these transcription factors using primary human Mj is sparsely described. Considering substantial differences between murine and human Mj, and the need for translational studies makes human Mj a relevant test system.
Our study aimed at describing activation of PPARs and LXRs in efferocytotic primary human Mj and explores how their activation shapes metabolism and inflammatory responses. The transcriptional LXR and PPAR responses in efferocytotic Mj demand lysosomal processing of ingested material and activities of LIPA and the lysosomal phospholipase A 2 .

Cell Culture and Reagents
Jurkat T cells were purchased from ATCC and maintained in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin and 100 mg/mL streptomycin. Human monocytes were isolated from commercially available buffy coats from anonymous donors (DRK-Blutspendedienst Baden-Württemberg -Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt, Germany) using Ficoll (Biochrom) density centrifugation. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using positive selection with CD14 antibody-coupled magnetic beads (MACS Miltenyi Biotec) and LS columns (MACS Miltenyi Biotec) following the manufacturer's protocol.

In Vitro Efferocytosis Assay
To induce apoptosis, Jurkat cells were seeded in 10 cm dishes in serum-free RPMI 1640 medium and exposed to 100 mJ/cm 2 UV-C (254nm) irradiation (UVP Crosslinker CL-1000, Jena Analytik) followed by incubation for 3 hours at 37°C with 5% CO 2 . Human Mj were stimulated with apoptotic Jurkat cells at a 1:3 ratio in 6-well plates for 3 or 6 hours. Upon efferocytosis, non-phagocytosed cells were removed and Mj were washed 3x with PBS, followed by incubation with RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin and 100 mg/mL streptomycin for 3, 6, 9 and 21 hours.

Apoptosis Analysis
Apoptosis of Jurkat cells was assessed with Annexin V-FITC/ propidium Iodide (PI) double staining. Upon UV treatment, Jurkat cells were washed with PBS and resuspended in Annexin V Binding Buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 2.5 mM CaCl 2 in PBS) with 1 µg/mL of Annexin V-FITC (Immunotools) and 1 µg/mL of PI (Thermofisher Scientific). Samples were incubated at room temperature in the dark for 15 minutes followed by flow cytometry analysis with a LSRII/ Fortessa flow cytometer (BD Biosciences).

Efferocytosis Analyses
Jurkat cells were labelled using CellTracker ™ Orange CMRA (ThermoFisher Scientific) according to the manufacturer's recommendations. Mj were seeded onto 8-well chambered coverslips (µ-slide, ibidi GmbH), stained with CFSE Cell Division Tracker Kit (Biolegend) and incubated with labelled apoptotic Jurkat cells at a 1:3 ratio. Upon removal of the nonphagocytosed cells, Mj were analyzed by fluorescence imaging using a Plan-Apochromat 20x long range objective on a Zeiss LSM800 confocal microscope driven by the Zen 2009 software (Carl Zeiss) or by flow cytometry with a LSRII/Fortessa flow cytometer (BD Biosciences).

NGS Library Preparation and RNA Sequencing
Total RNA of non-treated and efferocytotic Mj (6 biological replicates each) was isolated using RNeasy Micro Kit (Qiagen) according to the manufacturer's protocol. cDNA library preparation was carried out using QuantSeq 3' mRNA-Seq Library Prep Kit FWD from Illumina (Lexogen) according to the manufacturer's procedure. RNA and DNA quantification was done using Qubit cDNA HS Assay Kits (ThermoFisher Scientific) and quality control was performed using an Agilent 2100 Bioanalyzer with RNA Nano Chip (Agilent) as well as High Sensivity DNA chips (Agilent). Libraries were diluted and denatured according to the Illumina Denature and Dilute Libraries Guide, followed by mixing with 1% Phix Control (Illumina). 12 Libraries were loaded on one sequencing cartridge of the TG NextSeq 500/550 High Output Kit v2 (75 cycles) (Illumina) and RNA sequencing was performed on a NextSeq500 system (Illumina).

RNA-seq Data Processing, Differential Expression Analysis and GSEA Analysis
Statistics of the individual RNA sequence data sets were monitored by FastQC analysis. RNA-seq data processing and differential expression analysis was performed using the QuantSeq data analysis pipeline from Bluebee Genomics analysis platform following manufacturer's instructions. Genes significantly regulated by efferocytosis were extracted by setting a threshold based on the average number of reads to avoid Jurkat T cellspecific genes (set at the level of CD3). 39.555 genes with lower number of reads were removed. After this selection, a 20.646 gene set was further analyzed using Gene Set Enrichment Analysis 3.0. Gene list of up-and down-regulated genes of efferocytotic versus non-treated MF was generated using the following inclusion criteria: |log 2 FC|>1 and Padjusted≤0.05, and normalized base mean above 50. The lists were ranked based on adjusted P value. The RNA-seq data are available at the Gene Expression Omnibus database under accession number GSE169160.

RNA Extraction and Q-PCR
Total RNA was isolated with peqGOLD RNAPure reagent (PeqLab Biotechnology) according to manufacturer's recommendations followed by reverse transcription using Maxima first-strand cDNA synthesis kit (ThermoFisher Scientific). Quantitative realtime PCR (Q-PCR) assays were performed with PowerUp SYBR Green Master Mix (Applied Biosystems) using Quant Studio Real Time PCR System (Applied Biosystems). Relative transcript amounts were quantified using the Dct method with bmicroglobulin (bMG) as a housekeeping gene and normalized to the untreated or apoptotic cell-treated controls.

Cholesterol Efflux Measurements
Human Mj were stimulated with apoptotic Jurkat cells at a 1:3 ratio in 6-well plates for 3 hours. Upon efferocytosis, nonphagocytosed cells were removed and Mj were washed 3 times with PBS, followed by incubation with phenol redfree RPMI 1640 supplemented with 10 µg/mL of human recombinant ApoAI (Calbiochem), 100 U/mL penicillin and 100 mg/mL streptomycin for 21 hours. Cholesterol levels in the supernatant of efferocytotic Mj were measured using the Amplex Red Cholesterol Assay Kit (Invitrogen) following the manufacturer's protocol.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.2. Data were analyzed using Student unpaired, two-tailed t test or by one-way ANOVA with Bonferoni multiple comparisons. Comparisons to normalized controls were analyzed using onesample t test. Graphical data are presented as means ± SEM for at least three independent experiments. Asterisks indicate significant differences between experimental groups (*p<0.05, **p<0.01, ***p<0.005).

LXR and PPAR Target Gene Induction Following Efferocytosis
PPARs and LXRs are well-established master transcriptional regulators of efferocytotic Mj, although their functional input to shape the phenotype of human Mj is only poorly understood. To follow LXR/PPAR activation, we set up an in vitro human efferocytosis model, consisting of monocyte-derived human Mj and apoptotic Jurkat T cells ( Figure 1A). Apoptosis of Jurkat cells was induced by UV-C exposure, which caused 75-85% apoptotic cells after 3 hours. To confirm time-dependent efferocytosis, CMRA Orange-labelled AC were added to Mj for 3 hours, followed by the removal of the non-phagocytosed cells, and subsequent incubations in fresh medium for up to 24 hours. Confocal microscopy showed efficient efferocytosis after 3 and 6 hours when Mj engulfed substantial amounts of apoptotic material ( Figure 1B). Quantification of these results by flow cytometry revealed roughly 60% CFSE + /CMRA Orange + Mj after 3 hours ( Figure 1C). Using microscopy or flow cytometry we noticed a decreasing fluorescence signal intensity originating from AC, suggesting that Mj digest the apoptotic material over time. We then analyzed the mRNA expression of classical LXR target genes, i.e., ABCA1 and ABCG1 as well as PPARd/g targets pyruvate dehydrogenase kinase 4 (PDK4) and carnitine palmitoyltransferase 1A (CPT1A) ( Figure 1D). Expression of LXR and PPAR mRNA targets peaked at 6-9 hours post-efferocytosis and returned to baseline after 24 hours. Therefore, we choose 6 hours (3 hours exposure to AC, followed by 3 hours after their removal) for subsequent gene expression analyses. Q-PCR analyses of genes specifically expressed in Jurkat cells (CD3E, CD3D, LCK) revealed that the amount of Jurkat mRNA did not exceed 1% of total mRNA in the samples. Furthermore, Jurkat cells showed negligible ABCA1 and PDK4 mRNA expression as compared to macrophages. Therefore, we excluded significant contribution of RNA from apoptotic cells in Mj. Data so far confirm rapid activation of PPAR and LXR target genes in efferocytotic human Mj.

RNA-Seq Analysis of Efferocytotic Human Mj
To explore global transcriptional responses of efferocytotic human Mj we performed RNA-seq analysis using 6 biological replicates. Again, Mj were exposed to AC for 3 hours, followed by AC removal and subsequent incubations for 3 hours. To group differentially expressed, functional subsets of genes, we performed gene set enrichment analysis (GSEA). Efferocytotic Mj exhibited transcriptional changes related to several metabolic and stress-signaling pathways. We noticed upregulation of oxidative stress responses and hypoxia pathways, as previously reported by us and others (15-17) (Figure 2A). Moreover, in addition to upregulating genes referring to PPAR and LXR pathways, GSEA data revealed strong downregulation of SREBP-2 targets associated with

Activation of LXR and PPARd Targets in Efferocytotic Mj
LXR and PPAR transcription factor families consist of two (LXRa and LXRb), respectively three (PPARa, PPARg, and PPARd) members. In human Mj LXRa is predominantly expressed (18) and linked to efferocytotic responses, while both PPARg and PPARd are equally expressed and are known to regulate efferocytosis-induced gene expression in various experimental systems. To understand the relative contribution of PPARd, PPARg and LXRs to individual target gene expression during efferocytosis, we inhibited PPARd and LXRs during the uptake of apoptotic Jurkat cells with the PPARd antagonist GSK3787 (19) or the LXR antagonist GSK2033 (20) followed by mRNA expression analysis of ABCA1, ABCG1, PDK4, and CPT1A. GSK3787 prevented the induction of PDK4 and CPT1A in efferocytotic Mj, while expression of ABCA1 and ABCG1 was not affected ( Figure 3A). In contrast, GSK2033 suppressed ABCA1 and ABCG1 expression but not that of PDK4 or CPT1A ( Figure 3B). Strikingly, GSK2033 increased PDK4 expression in the absence of AC, suggesting that this inhibitor might activate PPARd. This appears rational as GSK2033 had been described as a ligand for multiple nuclear receptors (21) and considering that GSK3787 attenuated effects of GSK2033 (Supplementary Figure 1A). PDK4 is still induced by AC in the presence of GSK2033 ( Figure 3B). PDK4 mRNA is also induced by the synthetic PPARg ligand rosiglitazone (Supplementary Figure 1B), but this induction is insensitive to GSK3787, confirming the specificity of the latter one as a PPARd antagonist. To further explore the contribution of PPARg and PPARd to the induction of PDK4, we silenced these transcription factors in efferocytotic Mj. Consistent with the repressive function of PPARd in the absence of a ligand (22), knocking down PPARd substantially upregulated PDK4 expression ( Figure 3C). AC failed to induce PDK4 RNA in PPARd-silenced cells, confirming a PPARd-dependence of PDK4 induction in efferocytotic Mj. In contrast, silencing PPARg preserved a PDK4 mRNA increase in

Lysosomal Phospholipase PLA2G15 Generates PPARd Ligands in Efferocytotic Mj
Currently, activation of PPARs and LXRs upon efferocytosis is only incompletely understood. We hypothesized that fatty acids and sterols, liberated upon lysosomal digestion of ingested material, might cause their transcriptional activation. To address this possibility, we inhibited lysosomal acidification using the lysosomal v-ATPase inhibitor concanamycin A. Blocking lysosomal acidification interfered not only with induction of LXR targets ABCA1 and ABCG1, but also the PPARd targets PDK4 and CPT1A ( Figure 4A). Conclusively, lysosomal processing of apoptotic cells is a pre-requisite for LXR and PPARd activation. Recently, Viaud and colleagues (14) suggested lysosomal acid lipase (LIPA) as an essential component to generate LXR ligands in efferocytotic THP-1 cells. To explore the relevance of this pathway for primary human Mj we silenced LIPA with over 90% efficiency both at the mRNA and protein level (Figures 4B, C). In agreement with published observations (14), LIPA silencing prevented induction of the LXR target ABCA1 mRNA by apoptotic cells. Strikingly, a LIPA knockdown also interfered with PPARd target gene expression in efferocytotic Mj ( Figure 4B). Thus, LIPA is critical for processing ingested material to generate both LXR and PPARd ligands. Also, the hydrolysis of ingested phospholipids by lysosomal phospholipase A 2 (PLA2G15) may generate PPAR activating ligands. To inhibit PLA2G15, we used isopropyl dodecylfluorophosphonate (IDFP), a covalent modifier of the catalytic serine-165 residue (24). As seen in Figure 4D, IDFP lowered PDK4 and CPT1A expression in efferocytotic MF, with no regulation of ABCA1 or ABCG1. To verify the role of PLA2G15, we used a knockdown strategy and analyzed mRNA expression of PPARd targets in efferocytotic Mj ( Figure 4E). Silencing PLA2G15 in Mj indeed attenuated PDK4 and CPT1A expression.

Antagonizing LXR Attenuates the ABCA1-Facilitated Cholesterol Efflux From Efferocytotic Mj
Previous studies suggested that LXRs and PPARs play important roles in controlling the expression of efferocytotic receptors, hours and subsequently exposed to AC for 6 hours. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. cholesterol efflux, and suppressing inflammatory responses in efferocytotic Mj (8,25). However, most of these studies employed Mj with a constitutive knockout of these transcription factors, with an altered expression of efferocytotic receptors or cholesterol transporters. We took advantage of acute pharmacological inhibition of LXRs and PPARd during efferocytosis to examine how LXR and PPARd activation in efferocytotic macrophages affects cholesterol efflux, mitochondrial metabolism, and inflammatory response. Surprisingly, and contrary to mRNA expression data, we noticed that protein expression of ABCA1 ( Figure 5A) was not significantly altered during efferocytosis. Analysis of Jurkatspecific protein Zap70 showed no contamination of the samples with proteins from engulfed cells. Nevertheless, when we analyzed cholesterol efflux to apoAI in the supernatant of Mj 24 hours post-efferocytosis ( Figure 5B), we observed an increased cholesterol accumulation in apoAI-containing medium. This increase is sensitive to the co-treatment with GSK2033 or the ABCA1 inhibitor probucol. Apparently, pharmacological LXR inhibition acutely suppresses ABCA1mediated cholesterol efflux from efferocytes.
PDK4 and CPT1A support mitochondrial fatty acid oxidation through inhibition of the mitochondrial pyruvate dehydrogenase complex (PDH) and maintaining fatty acid transport into mitochondria, respectively. While we were unable to find commercial antibodies specifically detecting PDK4 in human Mj, we did not observe changes in PDH phosphorylation upon efferocytosis (Supplementary Figure 2A). Similarly, CPT1A p r o t e i n r e m a i n e d u n a l t e r e d i n e ff e r o c y t o t i c M j (Supplementary Figure 2A). Finally, analysis of mitochondrial oxygen consumption and extracellular acidification rates using Seahorse extracellular flux analysis showed no alterations in efferocytotic Mj, suggesting that efferocytosis doesn't significantly affect mitochondrial metabolism in human Mj (Supplementary Figure 2B).
To assess the importance of LXR and PPARd in modulating the inflammatory profile of efferocytotic Mj, we acutely inhibited these transcription factors using GSK2033 and GSK3787, respectively, and simultaneously treated Mj with lipopolysaccharide (LPS) and apoptotic cells ( Figure 5C). LPS stimulated the mRNA expression of inflammatory chemokines, i.e., chemokine (C-X-C motif) ligand 9 and 10 (CXCL9 and CXCL10) as well as the pro-inflammatory interleukin 12A (IL12A). In efferocytotic Mj expression of CXCL9/10 and IL12A mRNA was attenuated. Whereas PPARd inhibition by GSK3787 attenuated the expression of these targets in LPSstimulated Mj, apoptotic cells still exhibited anti-inflammatory effects in the presence of GSK2033 or GSK3787. We did not observe significant alterations of anti-inflammatory IL10 in efferocytotic Mj under these conditions (Supplementary Figure 2C). In summary, these observations indicate that apoptotic cells attenuate LPS-induced inflammatory gene expression probably independently of LXR or PPARd agonsim.

DISCUSSION
In this study, we characterized changes in gene expression using a human efferocytosis model of primary Mj phagocytosing apoptotic Jurkat cells. RNA sequencing of efferocytotic Mj revealed transcriptional changes, referring to various metabolic and stress-signaling pathways that previously had been noticed by us and others (15)(16)(17). Thus, efferocytotic Mj upregulated glucose transporters SLC2A1 and SLC2A3 as well as other glycolytic genes, likely a consequence of hypoxia-induced factor 1a activation (15). Furthermore, we noticed upregulation of heme oxygenase-1, confirming our previous observations (26), as well as induction of a p53 transcriptional target CDKN1A. These stress responses may attenuate inflammatory activation of Mj upon apoptotic cell engulfment (26). Our data also show that LXRs, PPARs and SREBPs are the main transcriptional regulators of lipid metabolism in efferocytotic human Mj. A strong downregulation of SREBP-2 targets was previously observed in efferocytotic LR73 hamster phagocytes (17), and in human efferocytotic Mj (27). In human Mj, accumulation of sterol biosynthetic intermediates upon efferocytosis likely contributes to LXR activation and SREBP-2 suppression (27). In addition to sterol homeostasis targeted by LXRs and SREBP-2, fatty acid metabolism undergoes transcriptional regulation in efferocytotic Mj through PPARs. Interestingly, pharmacological and genetic interventions of PPARd suggested that this transcription factor is dominant in activating lipid metabolic genes upon efferocytosis, while PPARg targets do not appear to contribute. Obviously, PPARd is the main PPAR target during efferocytosis in the human setting.
Mechanisms how PPARs and LXRs are activated upon efferocytosis are not completely understood. Our data suggest that lysosomal processing of apoptotic cells is a pre-requisite for LXR and PPAR activation. Whereas the activity of LIPA was essential for generating LXR ligands in efferocytotic THP-1 cells (14), in our model the knockdown of LIPA suppressed induction of PPARd as well as LXR target genes. These findings are surprising, considering the LIPA shows some specificity towards digestion of neutral lipids, such as cholesterol esters and triglycerides, which are not the predominant lipid species in efferocytosed Jurkat cells. Since LIPA inhibition did not impair lysosomal acidification and proteolytic activity (14), we did not expect a general defect of lysosomal function in LIPA-silenced Mj. The exact mechanism how LIPA generates PPARd/LXR ligands thus remains enigmatic and necessitates further research.
Besides LIPA, we showed that pharmacological inhibition or a knockdown of PLA2G15 abolished induction of PPARd target genes. Conclusively, PPARd activation in efferocytotic Mj demanded the processing of engulfed phospholipids with the release of free fatty acids by lysosomal PLA2G15. These observations point to a pivotal role of lysosomal digestion in PPARd activation during AC clearance. Nonetheless, additional studies are necessary to explore the nature of lysosome-derived ligands necessary for PPAR and LXR activation.
LXR regulates cholesterol efflux to apoAI-containing medium (28,29). Our observations suggest that pharmacological LXR inhibition in Mj acutely suppresses ABCA1-mediated cholesterol efflux. Although we could not observe upregulation of total ABCA1 protein, LXR might enhance the ABCA1 transporting capacity in efferocytotic Mj, perhaps by specifically increasing the expression of the efflux-promoting pool of ABCA1 protein.
The PDH complex is responsible for adjusting the metabolic flexibility in mammals (30). PDH activity is suppressed through phosphorylation, catalyzed by four highly specific PDK isozymes (30). The most widely expressed are PDK2 and PDK4 in heart, liver and kidney of humans and rodents (30). Inactivation of PDH by upregulating PDK4 is known to shift glucose catabolism to fatty acid utilization (30,31). Here, we show the PPARd target PDK4 to be one of the strongest upregulated gene upon efferocytosis. Nevertheless, we failed to observe metabolic changes as described before (30,31), and moreover, could not detect an altered PDH phosphorylation. Additionally, PDH phosphorylation in Mj was insensitive to PDK4 silencing or upregulation by the PPARd agonist GW501516, but was blocked by the PDK inhibitor dichloroacetate (Supplementary Figure 3). Conclusively, PDK4 is not a major regulator of PDH phosphorylation in human macrophages.
Besides regulating AC uptake, LXR and PPARs are also implicated to attenuate inflammatory responses during efferocytosis (25,32). In LXR-deficient Mj the production of anti-inflammatory cytokines, i.e. TGFb or IL-10 upon AC engulfment as compared to the wild-type Mj is impaired (25). A LXR deficiency also failed to suppress LPS-induced IL-1b and IL-12 expression by AC. Similarly, PPARd-deficient efferocytotic Mj displayed reduced IL-10 and elevated TNFa and IL-12 secretion upon LPS-stimulation (8). In our experimental setting AC still attenuated LPS-induced inflammatory gene expression, even when LXR and PPARd were inhibited. This may reflect the lack of IL-10 and TGFb induction by AC under our conditions as well as differences between human and the mouse system. Our observations indicate that AC are able to attenuate LPS-induced inflammatory gene expression probably independently of LXR or PPARd agonism.
In summary, our findings present novel mechanistic insights on LXR and PPARd activation in human efferocytotic Mj. At the same time, our work highlights notable differences in the impact of PPARd and LXR inhibition on the metabolism and antiinflammatory phenotype of efferocytes as compared to rodent knockout models that deserve more attention and clarification in the future research.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession can be found below: https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE169160.

AUTHOR CONTRIBUTIONS
AM conceived and performed the experiments, analyzed the data, and drafted the manuscript. MD, AW, and RS contributed to data analysis and interpretation. DN and BB participated in study design, data analysis, and writing of the final manuscript draft. All authors contributed to the article and approved the submitted version.