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Published in final edited form as: Biochim Biophys Acta Mol Cell Biol Lipids. 2023 Feb 6;1868(4):159291. doi: 10.1016/j.bbalip.2023.159291

Functional Characterization of Interleukin 4 and Retinoic Acid Signaling Crosstalk During Alternative Macrophage Activation

Ivan Pinos a, Jianshi Yu b, Nageswara Pilli b, Maureen A Kane b, Jaume Amengual a,c,*
PMCID: PMC9974901  NIHMSID: NIHMS1873465  PMID: 36754230

Abstract

Retinoic acid possesses potent immunomodulatory properties in various cell types, including macrophages. In this study, we first investigated the effects at the transcriptional and functional levels of exogenous retinoic acid in murine bone marrow-derived macrophages (BMDMs) in the presence or absence of interleukin 4 (IL4), a cytokine with potent anti-inflammatory properties. We examined the effect of IL4 on vitamin A homeostasis in macrophages by quantifying retinoid synthesis and secretion. Our RNAseq data show that exogenous retinoic acid synergizes with IL4 to regulate anti-inflammatory pathways such as oxidative phosphorylation and phagocytosis. Efferocytosis and lysosomal degradation assays validated gene expression changes at the functional level. IL4 treatment altered the expression of several genes involved in vitamin A transport and conversion to retinoic acid. Radiolabeling experiments and mass spectrometry assays revealed that IL4 stimulates retinoic acid production and secretion in a signal transducer and activator of transcription 6 (STAT6)-dependent manner. In summary, our studies highlight the key role of exogenous and endogenous retinoic acid in shaping the anti-inflammatory response of macrophages.

INTRODUCTION

By modulating the expression of over 500 genes, retinoic acid, the transcriptionally active form of vitamin A, regulates energy metabolism and function of multiple cell types including macrophages (Balmer & Blomhoff, 2002). Retinoic acid signaling participates in both innate and adaptive immune responses such as myeloid cell differentiation, T cell development, and the production of intestinal gut-homing molecules in T and B cells (Bang et al., 2021; Kuwata et al., 2000; Miller et al., 2020; Raverdeau & Mills, 2014). These broad effects on immunity highlight the critical role of retinoic acid in modulating the immune response to infections and inflammation. For instance, vitamin A deficiency is associated with a greater susceptibility to infectious diseases and increased mortality in children, remaining a major public health problem in developing countries (Bailey et al., 2015; Mora et al., 2008; Sommer et al., 1986).

Alternative activation in macrophages (= anti-inflammatory or M2) has been associated with increased retinoic acid signaling (He et al., 2021), and studies using different macrophage models suggest that exogenous retinoic acid induces alternative activation (C. Chen et al., 2019; Feng et al., 2017; Ho et al., 2016; Vellozo et al., 2017). This alternative activation typically occurs in response to extracellular signaling cues such as interleukin 4 (IL4), an anti-inflammatory cytokine secreted by T helper 2 cells (Walker & McKenzie, 2018). Conversely, classical activation (or M1) is induced by pro-inflammatory signals such as lipopolysaccharide or interferon-gamma (Murray et al., 2014). Alternatively activated macrophages favor the resolution of inflammation and tissue repair, associated with a greater capacity to remove apoptotic cells through efferocytosis than naïve or pro-inflammatory macrophages (Gordon & Martinez, 2010; Korns et al., 2011).

The terminal reaction in retinoic acid synthesis is catalyzed by three retinal dehydrogenases (RALDH1–3), which are encoded by three separate genes Aldh1a1, Aldh1a2, and Aldh1a3 (Belyaeva et al., 2019; S. Wang et al., 2020). During the embryo development, the spatiotemporal regulation of these enzymes ensures the correct formation of organs and tissues, but the stimuli that regulate their expression in adult organs and cells remain elusive (Guadix et al., 2011; Niederreither, Fraulob, et al., 2002; Niederreither, Vermot, et al., 2002). Various experimental models show that alternatively activated macrophages and dendritic cells over-express Aldh1a2, suggesting that these cells can synthesize and secrete retinoic acid (Broadhurst et al., 2012; Gundra et al., 2014; Nagy et al., 2012; Ohoka et al., 2014; Rajakumar et al., 2020; Zhu et al., 2013). However, whether macrophage alternative activation results in the net production of retinoic acid has not been assessed to date.

In this study, we first sought to investigate the role that exogenous retinoic acid plays in macrophages, to later establish the effect of anti-inflammatory status has on endogenous retinoic acid synthesis in these cells. To this end, we utilized a standardized cell culture system consisting of murine bone marrow-derived macrophages (BMDMs) exposed to IL4, widely utilized to polarize naïve macrophages into anti-inflammatory macrophages (Murray et al., 2014). By combining transcriptomics with functional assays, we establish the effect that exogenous retinoic acid has on naïve versus IL4-stimulated BMDMs. We also performed HPLC, mass spectrometry, and radiolabeling studies to quantify the intracellular production and secretion of vitamin A, either as retinoic acid or as retinol bound the retinol-binding protein 4 (RBP4), in naïve and IL4-treated macrophages.

MATERIALS AND METHODS

Isolation and differentiation of BMDMs

All procedures and experiments were performed in compliance with the Institutional Animal Care and Use Committee of the University of Illinois at Urbana Champaign. Bone marrow isolation and macrophage differentiation were performed following established protocols (Toda et al., 2021; Ying et al., 2013). Briefly, 6–12 weeks old male and female C57BL/6J wild-type (#000664, Jackson Laboratory, Bar Harbor, ME), Stat6−/− (#005977, Jackson Laboratory), and 129XC57BL/6J Rbp4−/− and wild-type controls (Quadro, 1999) were euthanized using CO2 and hind limbs dissected at the level of the hip joint. Bone marrow cells were isolated under sterile conditions and cultured in non-adherent Petri dishes (Thermo Fisher Scientific, Waltham, MA) in a 5% CO2 atmosphere at 37°C for seven days in complete growth medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA), 100 U/mL penicillin (Corning, Corning, NY), 100 μg/mL streptomycin (Corning), and 1% L-Glutamine (Corning). For macrophage differentiation, complete growth medium was supplemented with 10 ng/mL murine macrophage colony-stimulating factor (M-CSF) (Gemini Bio Products, Sacramento, CA).

Macrophage activation and retinoic acid treatment

Fully differentiated BMDMs were washed with phosphate-buffered saline (PBS) and incubated for 15 minutes with TrypLE Express Enzyme (Gibco) to detach cells. Cells were counted and plated in 12-well plates with complete growth medium. Next day, we added fresh medium supplemented with 20 ng/mL IL4 (Gemini Bio Products) for 24 hours or the same medium without IL4 (control medium). Cells were then washed with PBS and treated for six hours with either 1 μM all-trans retinoic acid (hence, retinoic acid or RA) (Sigma-Aldrich, St. Louis, MO) dissolved in dimethyl sulfoxide (DMSO) or DMSO alone as vehicle control.

RNA isolation

After the corresponding treatments, we washed the cells with PBS and added TRI reagent (Zymo Research, Irvine, CA). RNA was isolated using Direct-zol RNA Microprep kit (Zymo Research) according to the manufacturer’s instructions, including DNase I treatment to prevent DNA contamination. RNA concentration was quantified using NanoDrop 2000 (Thermo Fisher Scientific) and its integrity was assessed using 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA).

Real-Time qPCR analyses

RNA isolated samples were reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using TaqMan Fast Advanced Master Mix or PowerUp SYBR Green Master Mix (Applied Biosystems) and the following TaqMan probes (Applied Biosystems) and primers (Integrated DNA Technologies, Coralville, IA): aldehyde dehydrogenase family 1 subfamily A2 (Aldh1a2; Mm00501306_m1), retinol-binding protein 4 (Rbp4; Mm00803264_g1), retinoic acid receptor beta (Rarb; Mm01319677_m1), hypoxanthine-guanine phosphoribosyl transferase (Hprt; Mm03024075_m1), dehydrogenase/reductase (SDR family) member 9 (Dhrs9; 5’-CAGTGGGTGAAGAGCCATGT-3’ and 5’-CAGTCAGTGGGAGCCAACA-3’), and tubulin beta 1 class VI (Tubb1; 5’-CAGGGCTTCTTGGTTTTCC-3’ and 5’-GGTGGTGTGGGTGGTGAG-3’). Gene expression analyses were performed with the StepOnePlus Real-Time PCR System (Applied Biosystems) and the ΔΔCt calculation method using Hprt and tubulin as housekeeping genes.

RNA sequencing

RNA sequencing was performed using RNA isolated from fully differentiated BMDMs isolated from male mice. The RNAseq libraries were prepared with TruSeq Stranded mRNAseq Sample Prep kit (Illumina, San Diego, CA), quantitated by qPCR, and sequenced on one SP lane for 101 cycles from one end of the fragments on a NovaSeq 6000 System (Illumina). Fastq files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software (Illumina), which also trimmed adapters. Salmon (Patro et al., 2017) (v1.4.0) was used to quasi-map reads of the NCBI Mus musculus Annotation Release 109 transcriptome using the entire GRCm39 genome as the decoy sequence and additional options (--seqBias --gcBias --numBootstraps=30 --validateMappings). Gene-level counts were then estimated based on transcript-level counts using the “bias corrected counts without an offset” method from the tximport package (Soneson et al., 2015). Gene-level counts were normalized using TMM method (Robinson & Oshlack, 2010) and genes without at least 0.5 counts per million in at least 7 samples were filtered out.

Gene set enrichment analysis

Pathway enrichment analyses were performed using the Gene Set Enrichment Analysis (GSEA) software v4.2.2 (UC San Diego, CA; Broad Institute, MA). This threshold-free approach analyzes all available genes from a dataset without prior filtering and it is recommended for bulk RNAseq data analyses (Reimand et al., 2019). Briefly, GSEA ranks genes on a dataset according to their differential expression between groups and calculates the degree of overrepresentation or enrichment score (ES) of a defined set of genes at the top/bottom of the given ranked dataset. The ES is normalized by accounting for the gene set size to obtain a normalized enrichment score (NES), and the level of significance is estimated using phenotype-based permutation tests followed by multiple testing correction (Subramanian et al., 2005). Curated gene sets used in the present study included the collection of “Hallmark gene sets” and the “Reactome” database from the Molecular Signatures Database (MSigDB v7.4). An FDR cutoff <0.05 was established to identify significantly overrepresented gene sets (Reimand et al., 2019; Subramanian et al., 2005).

Efferocytosis assay

We evaluated the efferocytic capacity of fully differentiated BMDMs isolated from both male and female mice by co-culturing them with apoptotic Jurkat cells (Clone E6–1, ATCC, Manassas, VA) using an efferocytosis assay kit, following the manufacturer’s instructions (Cayman Chemical Company, Ann Arbor, MI). We grew Jurkat cells in Roswell Park Memorial Institute (RPMI) media (Sigma-Aldrich) containing 10% FBS (Gibco), 100 U/mL penicillin (Corning), 100 μg/mL streptomycin (Corning), and 1% L-Glutamine (Corning). Before the efferocytosis assay, Jurkat cells were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) for 30 minutes followed by incubation with 1 μM staurosporine for four hours to induce apoptosis (Evans et al., 2017). Apoptotic CFSE-labeled Jurkat cells were added to plates containing BMDMs at a ratio of 1:1 and co-cultured for 30 minutes. Non-phagocyted apoptotic cells were removed with cold PBS before harvest.

Flow cytometry

BMDMs isolated from both male and female mice were suspended in flow-activated cell sorting (FACS) buffer (0.5% BSA in PBS) and incubated with Pacific Blue anti-mouse CD45 (clone 30F11, BioLegend, San Diego, CA) and APC-Cyanine7 anti-mouse F4/80 (clone AFS98, BioLegend) for 30 minutes on ice. Viable cells were selected using Sytox Red Dead Cell Stain (Invitrogen, Waltham, MA). Data were collected and analyzed using BD LSR II Flow Cytometry Analyzer (BD Biosciences, Franklin Lakes, NJ) and FACS Diva Software v.8.0.1 (BD Biosciences), respectively.

Long-lived protein breakdown rate assay

Protein breakdown rate was examined following a protocol described by Zhao and Goldberg (Sha et al., 2018). Briefly, BMDMs isolated from both male and female mice were first radiolabeled by an overnight incubation with complete growth medium supplemented with 5 μCi/mL [3H]-Phenylalanine ([3H]-Phe) (American Radiolabeled Chemicals, St, Louis, MO). Cells were then washed and subsequently incubated for two hours with complete growth medium containing 2 mM non-radioactive Phe (Sigma-Aldrich). Two hours later, cells were washed and pre-treated for two hours with chase medium containing the corresponding treatments: 100 nM torin 1 (Sigma-Aldrich), 100 nM bafilomycin A1 (Sigma-Aldrich), 1 μM retinoic acid, or DMSO as vehicle. After pre-treatment, 200 μL media were sequentially collected every hour for six hours. After the last time point, remaining medium was discarded, and cells were lysed by adding 0.2 M NaOH. Protein fraction from the media was precipitated by incubation with 20 μL trichloroacetic acid (Sigma-Aldrich) for 30 minutes, followed by centrifugation at 20,000xg at 4°C for 15 minutes. Supernatants and cell lysates were transferred to 3 mL vials containing Bio-Safe NA counting cocktail (Research Products International, Mt. Prospect, IL) and analyzed in an LS6500 Scintillation System (Beckman Coulter, Brea, CA). The cumulative [3H] counts per million (cpm) released each hour was divided by the total cpm obtained in both the media and the lysates to calculate the percentage of cell protein degraded per hour. Degradation rate via lysosomal pathway was calculated by subtracting the degradation rate in bafilomycin A1-treated samples from the overall breakdown rate, as described (Sha et al., 2018).

Autophagy flux assay

Autophagy flux was analyzed as done in the past (Amengual, Guo, et al., 2018). Briefly, BMDMs isolated from both male and female mice were polarized and subsequently exposed to 1 μM retinoic acid or DMSO for six hours as previously described. Autophagy flux in BMDMs was blocked by incubating with the protease inhibitors (P.I.) E64d (30 μM) and leupeptin (100 μM) (Sigma-Aldrich). Cells were harvested and total protein was extracted using 2x Laemmli Sample Buffer (Bio-Rad, Hercules, CA) containing 2-mercaptoethanol (Bio-Rad).

Determination of intracellular and extracellular retinoids

BMDMs isolated from both male and female mice were treated as outlined above. For these experiments, we utilized wild-type, Stat6−/−, and Rbp4−/− mice. Cells were washed with cold PBS, detached using TrypLE Express Enzyme, and pelleted by centrifugation at 500 xg for five minutes at 4°C. Cells and media were assayed for retinoids separately. For cells, individual samples consisted of 5.3 ± 2.7 million cells (range: 1.4 – 13.5 million cells) with n=3–6 biological replicates for each condition. Cells were lysed in 0.8 mL of saline and 0.7 mL was extracted. For media, an aliquot of 0.8 mL of media was extracted from a total media volume of 9.0 ± 0.8 mL (range: 8–10 mL) for each sample with n=3–6 biological replicates for each condition. Retinoids were extracted using a two-step liquid-liquid extraction under yellow UV-blocking lights as previously described in detail (Kane & Napoli, 2010; Yu et al., 2022). The first step of the extraction yields retinol and retinyl esters, whereas the second step of the extraction yields retinoic acid. Retinol and total retinyl esters were quantified by HPLC-UV using a Water H-Class ACQUITY system as described previously (Kane et al., 2008; Kane & Napoli, 2010). Retinoic acid was quantified by liquid chromatography-multistage-tandem mass spectrometry using a Shimadzu Prominence UFCL XR liquid chromatography system (Shimadzu, Columbia, MD) coupled to an AB Sciex 6500 QTRAP hybrid triple quadrupole mass spectrometer (AB Sciex, Framingham, MA) using atmospheric pressure chemical ionization operated in positive ion mode as described previously (Jones et al., 2015). Intracellular retinoid content was normalized to the total cellular protein content in each sample and retinoid content in the media was normalized to the total cell culture media volume in each sample.

[3H]-Retinol radiolabeling and RBP4 immunoprecipitation

To examine the role of macrophage-derived RBP4 in vitamin A export, we gavaged three male mice with 100 μL of olive oil containing 100 μCi of [3H]-retinol (American Radiolabeled Chemicals) to extract their plasma for cell culture experiments. To avoid the interference of [3H]-retinol bound to RBP4, we utilized Rbp4−/− mice. Four hours after the gavage, mice were exsanguinated under sterile conditions to collect [3H]-retinyl ester-packed chylomicrons, as done in the past (Acharya et al., 2022). Sera were pooled, filtered 0.22 μm pore size syringe filters (Thermo Fisher Scientific), and frozen until use. To load macrophages with [3H]-vitamin A, we added the sera to the growth media two days prior to performing our treatments. After the complete differentiation, we plated the cells in FBS-free media without [3H]-vitamin A containing IL4 or not (control). Immunoprecipitations of RBP4 secreted to the media and cell lysates were performed by adapting our previous protocols (Amengual, Guo, et al., 2018). Briefly, cell media were collected, mixed with 5 μL of protease inhibitor cocktail (Thermo Fisher) and 5 μL of phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich), and then centrifuged at 1,000 xg for five minutes at 4°C to remove any cell debris. Cells were washed with cold PBS and lysed with 1 mL of 1xNET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 7.4) containing 60 mM sucrose, 0.05% Triton X-100, 0.5% sodium deoxycholate, and 5 μL of protease inhibitors and PMSF in an orbital shaker for 30 minutes on ice. RBP4 immunoprecipitation was carried out using 5 μg of rabbit anti-RBP4 (Agilent Technologies) and 50 μL of Protein A-Sepharose 4B (Invitrogen) in an orbital shaker at 4°C overnight. Next day, beads were washed with 1xNET buffer three times and then transferred to 3 mL vials containing Bio-Safe NA counting cocktail (Research Products International, Mt. Prospect, IL) for quantification of the [3H]-retinol bond to RBP4 in an LS6500 Scintillation System (Beckman Coulter, Brea, CA).

Immunoblotting

Protein extracts were fractioned on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto methanol-activated polyvinylidene difluoride (PVDF) membranes using a transfer unit according to manufacturer’s instructions (Bio-Rad). Membranes were blocked with 5% fat-free milk powder in Tris-buffered saline (15 mM NaCl and 10 mM Tris-HCl, pH 7.5) containing 0.01% Tween 100 (TBS-T) for one hour at room temperature. Membranes were washed and subsequently probed overnight at 4°C with primary antibodies diluted in TBS-T (1:1,000). Primary antibodies used in these experiments were rabbit anti-arginase 1 (clone 16001–1-AP, Proteintech, Rosemont, IL), rabbit anti-microtubule associated protein 1 light chain 3 alpha (LC3) (clone PM036, MBL International, Woburn, MA), rabbit anti-RBP4 (Agilent Technologies), and mouse anti-tubulin (clone AA2, Sigma-Aldrich). Membranes were washed and incubated with IRDye 650 anti-mouse or anti-rabbit secondary antibodies (Li-Cor Biotechnology, Lincoln, NE) prepared at 1:15,000 in TBS-T with 5% fat-free milk powder for one hour at room temperature. Blots were visualized in the ImageQuant LAS4000 instrument (HE Healthcare, Chicago, IL) and quantified using Image Studio Lite (v.5.2, Li-Cor Biotechnology).

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (v9.2.0) and results expressed as the mean ± standard error of the mean (SEM). Statistical tests were performed using ordinary one-way or two-way ANOVA using Tukey’s multiple comparisons test. D’Agostino-Pearson Omnibus test was used to assess for data normality and, when assumption of normality was not met, Kruskal-Wallis test with Dunn’s multiple comparison test was performed. A p-value <0.05 was considered statistically significant. For RNAseq, differential gene expression analysis was performed using the limma-trend method (Y. Chen et al., 2016) using a model of ~Trt + 4 quantitative factors estimated by RUVSeq (Risso et al., 2014) (v1.26.0) to remove spurious technical variation. Multiple testing correction was done using the false discovery rate (FDR) method (Benjamini & Hochberg, 1995) and a cutoff for FDR <0.05 was set for significance.

RESULTS

Retinoic acid exposure synergizes with the effect of IL4 in murine BMDMs

To explore the effect of IL4 and exogenous retinoic acid on macrophage gene expression, we performed RNAseq using BMDMs isolated from three a wild-type mice. Differential gene expression analyses showed that IL4-primed BMDMs presented twice as many genes regulated by retinoic acid than naïve macrophages (1,410 and 753, respectively). The retinoic acid-responsive genes Rarb and Cyp26b1 were upregulated in both naïve and IL4-stimulated macrophages (Figure 1A, B). Of those genes that responded to retinoic acid, 54% were exclusively regulated in IL4-treated cells whereas only 14% were exclusive in naïve BMDMs (Figure 1C). Multidimensional scaling analysis displayed a clear separation between naïve and IL4-treated cells, followed by a second dimension in which we observed the effect of retinoic acid exposure. Relative distance between the four experimental groups highlighted the differential effect of retinoic acid on macrophage gene expression depending on the pre-exposure to IL4 (Figure 1D).

FIGURE 1. Retinoic acid exposure synergizes with IL4 to regulate gene expression in murine bone marrow-derived macrophages (BMDMs).

FIGURE 1.

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Fully differentiated BMDMs from three age-matched male wild-type mice were cultured in normal growth medium (naïve) or the same medium supplemented with 20 ng/mL IL4 (IL4) for 24 hours. After this period, cells were exposed to 1 μM of retinoic acid (RA) or vehicle control (DMSO) for six hours prior to RNA isolation and sequencing. A) Volcano plot showing differentially expressed genes in naïve and B) IL4-treated macrophages in response to retinoic acid. C) Venn’s diagram showing the relative number of significantly regulated genes by retinoic acid in naïve and IL4-treated macrophages (FDR<0.05). D) Multidimensional scaling plot. E) Top-most regulated hallmark gene sets in IL4-treated macrophages in response to retinoic acid. F, G) Detailed GSEA output of the most upregulated (oxidative phosphorylation, OXPHOS) and downregulated (endothelial to mesenchymal transition, EndMT) gene sets. H) Heatmap representation of the top five genes regulated by IL4 across samples. I, J) Representative western blot and quantifications for arginase 1 and tubulin (loading control). NES: Normalized enrichment score; FDR: False discovery rate. Data represented as the mean ± SEM (n= 3 mice/group). Volcano plot analyses were performed using the lima-trend method followed by one-way ANOVA with pairwise comparisons. Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test. Means with different letters indicate statistically significant difference (P<0.05).

To examine phenotypical signatures on IL4-treated macrophages exposed to retinoic acid in comparison to our other experimental groups (naïve, vehicle group; naïve, retinoic acid group, and IL4, vehicle group), we performed GSEA analysis using Hallmark gene set collections from the MSigDB (Figure 1E). GSEA analysis indicated a generalized upregulation of genes implicated in oxidative phosphorylation (OXPHOS) in comparison to the other experimental groups, and a downregulation of genes involved in endothelial to mesenchymal transition (EndMT) (Figure 1F, G). Because OXPHOS is a hallmark of alternatively activated macrophages (Kelly & O’Neill, 2015; Wculek et al., 2022), while EndMT is a characteristic feature of pro-inflammatory macrophages (Ge et al., 2021), these results suggest that retinoic acid synergizes with IL4 to enhance an anti-inflammatory phenotype in macrophages, in agreement with previous reports (C. Chen et al., 2019, 2020; Feng et al., 2017; Ho et al., 2016; Vellozo et al., 2017).

To further explore the anti-inflammatory effect of retinoic acid in macrophages, we examined the expression of classical IL4-responsive genes in murine macrophages. To this end, we selected the top five genes with a greater fold change between the naïve and IL4 groups and compared their expression across our four experimental groups (Figure 1H). We noticed a generalized upregulation of these genes upon retinoic acid exposure in IL4-treated macrophages, with Arg1, a typical marker of alternative activation in murine macrophages (Murray et al., 2014), being the one of the most synergistically upregulated genes. These changes in mRNA levels were supported by the arginase 1 protein (Figure 1I, J), as previously reported under similar experimental conditions (Ho et al., 2016; Lee et al., 2016). Overall, these results show that the combination of IL4 and retinoic acid stimulates the expression of genes and pathways aligned with an anti-inflammatory phenotype.

Exogenous retinoic acid promotes efferocytosis and lysosomal degradation in IL4-stimulated macrophages

Given that changes in transcriptional regulation do not always translate into functional outcomes (Gebauer & Hentze, 2004; Maggi & Weber, 2013), we sought to determine whether exogenous retinoic acid modulates the phenotype of naïve and IL4-stimulated BMDMs. To narrow down the synergistic effects of retinoic acid and IL4 in BMDMs, we focused on those genes differentially regulated by retinoic acid in IL4-polarized cells.

We first performed a GSEA analysis using the Reactome database, which unveiled “Binding and uptake of ligands by scavenger receptors” as the most upregulated pathway in response to retinoic acid in IL4-primed BMDMs (Figure 2A, B). Scavenger receptors are involved in phagocytosis and clearance of apoptotic cells, a process overall known as efferocytosis and a phenotypic feature of alternatively activated macrophages (Canton et al., 2013; Penberthy & Ravichandran, 2016). We performed efferocytosis assays using the same experimental conditions outlined above and following established protocols (Evans et al., 2017) (Figure 2C, D). As expected, IL4-exposed BMDMs showed 30% greater efferocytosis capacity than naïve BMDMs. Retinoic acid failed to alter efferocytosis in naïve macrophages, but it synergized with IL4 to enhance the uptake of apoptotic cells by almost 50% compared to naïve cells (Figure 2E, F).

FIGURE 2. Retinoic acid enhances efferocytosis in IL4-exposed bone marrow-derived macrophages (BMDMs).

FIGURE 2.

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Fully differentiated BMDMs isolated from both male and female age-matched wild-type mice were cultured in normal growth medium (naïve) or the same medium supplemented with 20 ng/mL IL4 (IL4) for 24 hours. After this period, cells were exposed to 1 μM of retinoic acid (RA) or vehicle control (DMSO) for six hours prior to RNA isolation and sequencing. A) Top-most enriched gene sets in IL4-treated macrophages in response to retinoic acid using the Reactome collection database. B) Relative gene expression of the top-most regulated genes within the “Binding and uptake by scavengers receptors” pathway in IL4-treated macrophages in response to retinoic acid (*FDR<0.05; **FDR<0.005; ***FDR<0.0005). C) Schematics showing experimental design for efferocytosis assay (see methods for details). D) Representative confocal microscopy image showing differentiated macrophages (F4/80+, red) with engulfed apoptotic Jurkat cells (CFSE-labeled, green). Nuclei were stained with DAPI. E) Representative gating strategy used to analyze macrophage efferocytosis by flow cytometry, and F) quantification of the relative percentage of engulfed Jurkat cells. G) Simplified schematics of efferocytosis and lysosomal degradation. H) Overall degradation, and I) and lysosomal degradation rates under our experimental conditions. J) Representative western blot and K) quantification of the autophagic flux in IL4-exposed macrophages in response to retinoic acid (see methods for details). P.I.: protease inhibitors. Date represented as the mean ± SEM (n= 4–6 mice/group and condition). Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test. Means with different letters indicate statistically significant difference (P<0.05).

Lysosomal degradation facilitates the elimination of apoptotic cells engulfed by efferocytosis after the phagosome-lysosome fusion (Doran et al., 2020) (Figure 2G). Hence, we examined whether retinoic acid promotes lysosomal degradation in naïve and IL4-treated BMDMs. To this end, we performed pulse-chase experiments following established protocols, where we measured the degradation of long-lived proteins using radiolabeled [3H]-phenylalanine (Sha et al., 2018). To evaluate the contribution of the lysosomal system to overall cellular protein degradation, we ran parallel experiments using torin 1 and bafilomycin A1 as positive and negative regulators of protein breakdown, respectively (data not shown). Retinoic acid stimulated overall protein breakdown, especially in those BMDMs previously stimulated by IL4 (Figure 2H). When we estimated the contribution of the lysosomal degradation to overall protein breakdown, the combination of IL4 and retinoic acid favored lysosomal degradation compared to the remaining experimental conditions (Figure 2I).

Lastly, we examined whether retinoic acid favored autophagy in IL4-stimulated cells by quantifying the autophagy flux, as done in the past (Amengual, Guo, et al., 2018). Retinoic acid did not alter the autophagic flux in IL4-treated macrophages (Figure 2J, K). Overall our data show that IL4-treated macrophages exposed to exogenous retinoic acid possess a greater phagocytic and lysolitic capacities in comparison to IL4 exposed macrophages.

IL4-stimulated macrophages promote vitamin A mobilization and retinoic acid synthesis in a STAT6-dependent manner

IL4 stimulates RALDH2 synthesis and activity in murine macrophages (Broadhurst et al., 2012; Gundra et al., 2014). Indeed, Aldh1a2 appeared as one of the top five genes regulated by IL4 in our RNA-seq results (Figure 1H). We further explored our RNAseq data to examine whether IL4 modulated other genes implicated in vitamin A metabolism and signaling (Figure 3A). IL4 treatment regulated several vitamin A transporters, enzymes, and transcription factors involved in the metabolism of vitamin A (Figure 3B). Because the upregulation of Aldh1a2 has been proposed to promote retinoic acid synthesis in IL4-treated macrophages (Broadhurst et al., 2012; Ouimet et al., 2015), we hypothesized that those genes upregulated by exogenous retinoic acid would also be upregulated in IL4-stimulated cells. However, from the top five genes upregulated in response to exogenous retinoic acid, only Rarb and Hic1 were upregulated by both IL4 and retinoic in comparison to naïve BMDMs (Figure 3C).

FIGURE 3. IL4 exposure promotes retinoic acid synthesis in macrophages in a STAT6-dependent manner.

FIGURE 3.

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Bone marrow-derived macrophages (BMDMs) isolated from both male and female age-matched wild-type or Stat6−/− C57BL/6 mice were differentiated and cultured in normal growth medium (naïve) or the same medium supplemented with 20 ng/mL IL4 (IL4) for 24 hours. A) General schematics of the vitamin A uptake from lipoproteins, vitamin A metabolism and conversion to retinoic acid, and secretion of vitamin A as free retinol or retinoic acid. B) Heatmap of vitamin A-related genes and C) relative expression of retinoic acid-responsive genes from our RNAseq analysis. D) Relative mRNA levels determined by RT-qPCR in response to IL4 in wild-type and Stat6−/− mice. E) Relative abundance of intracellular retinoids determined by HPLC-UV (retinyl esters, retinol) or LC-MS/MS (retinoic acid) in naïve and IL4-treated BMDMs. F) Relative abundance of retinoic acid released to the media determined by LC-MS/MS. Data represented as the mean ± SEM (n= 3–6 mice/group). ROL: Retinol; RAL: Retinal; REs: Retinyl esters. Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test. Means with different letters indicate statistically significant difference (P<0.05).

IL4 mediates the majority of its effects by activating the (STAT6) pathway (Takeda et al., 1996). To examine the implication of STAT6 on vitamin A homeostasis in the macrophage, we isolated BMDMs from wild-type and Stat6−/− mice and exposed them to IL4. As previously reported, Aldh1a2 upregulation by IL4 was STAT6-dependent (Broadhurst et al., 2012). Expression levels of the vitamin A transporter Rbp4 and the retinoic acid-responsive genes Dhrs9 and Rarb followed the same pattern (Figure 3D). HPLC-UV measurements of non-polar retinoids revealed that IL4 exposure decreased retinyl ester levels in wild-type mice without affecting intracellular retinol stores in wild-type cells, but not in STAT6-deficient BMDMs (Figure 3E). Intracellular retinoic acid levels measured via LC-MS/MS in wild-type macrophages increased in response to IL4, but not in BMDMs isolated from Stat6−/− mice (Figure 3E). In alignment with the intracellular retinoid levels, results obtained from the cell media showed a slight increase in retinoic acid content from IL4-stimulated wild-type macrophages, that was abrogated in cell media from Stat6−/− mice (Figure 3F).

Among the IL4-responsive genes related to vitamin A transport, Rbp4 caught our attention. RBP4 is mainly synthesized by hepatocytes, where it binds retinol and distributes it through the bloodstream (Blaner, 1989; Zanotti & Berni, 2004). To examine the contribution of macrophage-derived RBP4 to vitamin A excretion, we quantified intracellular and extracellular RBP4 levels in naïve and IL4-exposed wild-type BMDMs and grown in a FBS-free media to avoid interference of RBP4 present in the FBS. Due to the low expression levels of RBP4, we chose to perform immunoprecipitation assays to detect RBP4 in our experimental samples. To this end, we first examined the suitability of our anti-RBP4 IgG to efficiently immunoprecipitate RBP4 (Figure 4A).

FIGURE 4. Contribution of RBP4 to vitamin A homeostasis in the macrophage.

FIGURE 4.

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Bone marrow-derived macrophages (BMDMs) isolated from both male and female age-matched wild-type or Rbp4−/− 129XC57BL/6J mice were differentiated and cultured in normal growth medium (naïve) or the same medium supplemented with 20 ng/mL IL4 (IL4) for 24 hours. A) Representative western blot showing a titration of anti-RBP4 IgG adding increasing concentrations of FBS media (1%, 2%, 5%, 10%, and 20%) which contains RBP4. Rabbit IgG isotype control and 5% input highlight the specificity and relative RBP4 content in the FBS media solution, respectively. B) Representative western blot and quantifications derived from immunoprecipitated RBP4 in cell lysates (left) and media (right) from FBS-free grown BMDMs upon IL4 incubation. C) The amount of [3H]-retinol bond to RBP4 released to the media was quantified upon RBP4 immunoprecipitation. D) Relative abundance of intracellular retinoids determined by HPLC-UV (retinyl esters, retinol) or LC-MS/MS (retinoic acid) in naïve and IL4-treated BMDMs. E) Relative abundance of retinoic acid released to the media determined by LC-MS/MS. Data represented as the mean ± SEM (n= 3 to 6 mice/group). Statistical analyses were performed by ordinary two-way ANOVA with Tukey’s multiple comparisons test. Means with different letters indicate statistically significant difference (P<0.05).

Next, we confirmed that the increase of Rbp4 mRNA levels in response to IL4 also resulted in increased protein levels (Figure 4B, left). However, we failed to detect differences in the amount of RBP4 released to the media in response to IL4 stimulation (Figure 4B, right). These results were confirmed by the quantification of RBP4 bond to radiolabeled retinol ([3H]-retinol; see methods for details). Naïve and IL4-stimulated macrophages showed no differences in the amount of the extracellular [3H]-retinol bound to RBP4 (Figure 4C).

Lastly, we tested whether the absence of RBP4 in the macrophage could affect vitamin A homeostasis. Both wild-type and RBP4-deficient BMDMs showed a trend toward decreasing in retinyl ester and retinol stores in response to IL4 (Figure 4D). IL4 increased intracellular and secreted retinoic acid levels in BMDMs, independently of their genotype (Figure 4D, E).

DISCUSSION

IL4 and exogenous retinoic acid are two potent anti-inflammatory agents, which can act synergistically in the macrophage. Additionally, it has been proposed that IL4 stimulates the production of endogenous retinoic acid by upregulating Aldh1a2 expression, the main enzyme implicated in retinoic acid synthesis in the macrophage (Broadhurst et al., 2012; Gundra et al., 2014). The goal of this study was to determine (1) whether the combination of IL4 and exogenous retinoic acid result in functional, quantifiable phenotypic alterations in macrophages and (2) to establish the net effect of IL4 on endogenous vitamin A homeostasis and retinoic acid production. Because the phenotype of cultured macrophages is typically defined based on changes in gene expression, we first approached these questions by performing RNAseq in murine BMDMs primed with IL4 and subsequently exposed to retinoic acid (see methodology for details). Our RNAseq data analyses recapitulated the synergistic effect of IL4 and exogenous retinoic acid that other investigators reported in the past (Ho et al., 2016; Lee et al., 2016). These analyses also revealed that IL4 alone altered the expression of several genes involved in retinoid metabolism, including Aldh1a2, highlighting the crosstalk between of IL4 signaling and vitamin A homeostasis.

A major pathway regulated by IL4 in the macrophage is OXPHOS, which allows macrophages to rely on fatty acids as their primary energy source (Huang et al., 2014; Kelly & O’Neill, 2015). The inhibition of OXPHOS is sufficient to compromise IL4-induced macrophage differentiation, highlighting the importance of the metabolic substrate in the macrophage phenotype (F. Wang et al., 2018). Work carried out by Palou’s group shows that retinoic acid favors lipolysis by stimulating mitochondrial proliferation and activity in several cell types such as hepatocytes, myocytes, and both white and brown adipocytes (Amengual et al., 2012; Amengual, García-Carrizo, et al., 2018; Mercader et al., 2006; Tourniaire et al., 2015). These observations were reproduced in rodents, where retinoic acid exposure favored the OXPHOS capacity in the liver, the muscle, and adipose tissues (Amengual et al., 2008, 2010; Mercader et al., 2006; B. Wang et al., 2017). These studies aligned with our results in which IL4 + retinoic acid promoted OXPHOS in BMDMs and highlighted the important role of the combined effect of these agents in the regulation of cellular phenotype, transdifferentiation, and immunometabolism (Figure 1E).

The engulfment of apoptotic cells by macrophages is indispensable to maintaining tissue homeostasis and resolving inflammation (Elliott et al., 2017). Efferocytosis is one of the most distinctive features of alternatively activated macrophages and is key to fostering fatty acid oxidation and OXPHOS, triggering anti-inflammatory reprogramming and resolution (Zhang et al., 2019). In our study, retinoic acid upregulated several receptors involved in efferocytosis (Figure 2B), resulting in an increased efferocytic capacity of IL4-exposed macrophages (Figure 2E, F). Similarly, Sarang et al. showed an upregulation of phagocytosis-related receptors, such as Stab2 and Timd4, in BMDMs upon retinoic acid exposure, accompanied by an increase in their efferocytic capacity in comparison to pro-inflammatory macrophages (Sarang et al., 2014). Interestingly, this group previously reported both in vitro and in vivo an upregulation of Aldh1a1 and Aldh1a2 in macrophages after engulfment of apoptotic cells (Garabuczi et al., 2013), suggesting a critical role of endogenous retinoic acid synthesis in the elimination of apoptotic cells.

A plethora of studies show that modulating the inflammatory state of the macrophage is an attractive therapeutic target for treating diseases such as atherosclerosis (recently reviewed in (Barrett, 2020). Macrophages found in atherosclerotic lesions have a variety of phenotypic characteristics. For example, developing lesions are characterized by macrophages displaying a pro-inflammatory phenotype, while regressing lesions are enriched with anti-inflammatory, tissue-repair macrophages (Feig et al., 2012). The switch of pro- to anti-inflammatory macrophages has been proposed as a therapeutic target to promote the regression of atherosclerosis, a process that also implicates other cell types such as regulatory T (Treg) cells (Moore et al., 2018). Tregs are enriched in regressing plaques, and their systemic depletion hampers atherosclerosis regression in murine models (Sharma et al., 2020). The upregulation of the transcription factor FoxP3 drives the differentiation of naïve T cells to Tregs, and studies suggest that upregulation of Aldh1a2 in macrophages is sufficient to promote retinoic acid synthesis, which could be released to the extracellular space and favor Treg differentiation (Broadhurst et al., 2012; Gundra et al., 2014, 2017; Ouimet et al., 2015). These studies are supported by co-culture experiments, in which IL4-treated BMDMs were cultivated in the presence of naïve T cells. However, the authors failed to unequivocally demonstrate the formation or release of retinoic acid by macrophages and inferred that retinal dehydrogenase activity was responsible for the formation and release of retinoic acid (Ouimet et al., 2015). Similar conclusions were by other authors upon observing an upregulation of Aldh1a2 expression in macrophages exposed to IL13 and IL5, which have a similar effect than IL4 in promoting an M2-like phenotype (Zhu et al., 2013).

Methodological limitations in RNAseq pathway analysis can lead to data misinterpretation because of the omission of annotated genes on those pathways (Reimand et al., 2019). This prompted us to examine the effect of IL4 on proteins implicated in vitamin A homeostasis in comparison to naïve BMDMs (Miller et al., 2020; Napoli, 2020). Based on their function, we observed that IL4 treatment favored the expression of genes that promote the formation of vitamin A uptake, intracellular mobilization, and retinoic acid formation (Stra6l, Lpl, Lipa, Rdh1, Dhrs9, Aldh2a1), while others would restrain retinoic acid synthesis (Rdh11, Dhrs3, Rbp4) (Figure 3B). Recently, the role of DHRS9 on cellular metabolism has been expanded beyond its role in retinoid homeostasis. Kedishvili’s group that show that Dhrs9 displays a potent activity towards oxylipins, implicating this enzyme in inflammatory response (Belyaeva et al., 2022). Indeed, DHRS9 expression is upregulated in human anti-inflammatory macrophages, in alignment with our results in BMDMs exposed to IL4 (Riquelme et al., 2017). Additionally, IL4 treatment resulted in the upregulation of only two of the top five genes upregulated by retinoic acid, while downregulating one of them, and not affecting the expression of the other two (Figure 3C). The sum of these discrepancies on gene expression levels led us to explore the net effect of IL4 stimulation on vitamin A homeostasis and retinoic acid production and secretion. Our HPLC/MS data show for the first time that IL4 promotes retinoic acid synthesis and release in BMDMs. These data are in agreement with an increase retinoic acid production in alternative macrophages isolated from human placenta, where RALDH activity was positively correlated with retinoic acid levels (Rajakumar et al., 2020).

An important gene implicated in vitamin A homeostasis is RBP4. RBP4 is primarily produced by the liver, and its secretion depends on intracellular retinol levels. For example, the liver accumulates RBP4 upon vitamin A deficiency, and retinol supplementation results in its rapid release from the hepatocyte in cell culture and animal models (Amengual et al., 2013; Quadro, 1999). This regulatory mechanism also operates in the choroid plexus, where choroidal RBP4 is released in the cerebrospinal fluid to deliver vitamin A to the brain (Amengual et al., 2014). However, the role of RBP4 in the macrophage remains unclear. RBP4 expression in macrophages was first reported in 2010, where the authors showed that pro-inflammatory signals such as lipopolysaccharides and the tumor necrosis factor alpha reduce RBP4 expression and release to the media (Broch et al., 2010). In 2017, RBP4 was detected in developing plaques of atherosclerotic lesions, but whether RBP4 was secreted by macrophages or taken up by phagocytosis was not clarified (Liu et al., 2017).

Our data show that IL4 exposure in BMDMs results in the upregulation of RBP4 at mRNA and protein levels, which prompted us to determine whether IL4 stimulation in the macrophage results in the release of intracellular retinol. Our radiolabeling experiments show that the upregulation of RBP4 by the IL4-STAT6 axis is not accompanied by the release of RBP4 to the extracellular media. Further research is necessary to fully understand the role of RBP4 in macrophage vitamin A homeostasis.

In summary, we demonstrate that the synergistic effect of exogenous retinoic acid and IL4 results in functional phenotypic alterations in BMDMs. In addition, we report for the first time an increased mobilization of macrophage vitamin A stores to synthesize and secrete retinoic acid in response to IL4. The mechanism(s) by which retinoic acid is secreted, however, remain to be explored.

ACKNOWLEDGMENTS

The authors thank the Roy J. Carver Biotechnology Center at the UIUC for their services with both RNA sequencing and analysis, and Dr. Loredana Quadro (Rutgers University) for sharing the Rbp4−/− mice.

This work was supported by the National Institutes of Health (R01HL147252 to JA) and the University of Maryland, School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014 to MAK).

Footnotes

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CONFLICT OF INTEREST

The authors listed on this study declare no conflicts of interests.

DATA AVAILABILITY

The RNAseq data that support the findings of this study are openly available in (TBD) Additional data are available on request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The RNAseq data that support the findings of this study are openly available in (TBD) Additional data are available on request from the corresponding author.

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