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• W2979586864 abstract "Article10 October 2019Open Access Source Data Lipid droplet-dependent fatty acid metabolism controls the immune suppressive phenotype of tumor-associated macrophages Hao Wu Hao Wu The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Yijie Han Yijie Han University of Chinese Academy of Sciences, Beijing, China Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yasmina Rodriguez Sillke Yasmina Rodriguez Sillke Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany Search for more papers by this author Hongzhang Deng Hongzhang Deng Department of Polymer Science and Engineering, Key Laboratory of Systems, Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Search for more papers by this author Sophiya Siddiqui Sophiya Siddiqui Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Christoph Treese Christoph Treese Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Franziska Schmidt Franziska Schmidt Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Marie Friedrich Marie Friedrich Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Jacqueline Keye Jacqueline Keye Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Jiajia Wan Jiajia Wan The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China Search for more papers by this author Yue Qin Yue Qin orcid.org/0000-0001-9793-7964 National Center for Nanoscience and Technology, Beijing, China Search for more papers by this author Anja A Kühl Anja A Kühl iPATH.Berlin – Core Unit of the Charité, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Zhihai Qin Corresponding Author Zhihai Qin [email protected] The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China Search for more papers by this author Britta Siegmund Britta Siegmund Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Rainer Glauben Corresponding Author Rainer Glauben [email protected] orcid.org/0000-0003-2889-2525 Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Hao Wu Hao Wu The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Yijie Han Yijie Han University of Chinese Academy of Sciences, Beijing, China Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yasmina Rodriguez Sillke Yasmina Rodriguez Sillke Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany Search for more papers by this author Hongzhang Deng Hongzhang Deng Department of Polymer Science and Engineering, Key Laboratory of Systems, Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Search for more papers by this author Sophiya Siddiqui Sophiya Siddiqui Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Christoph Treese Christoph Treese Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Franziska Schmidt Franziska Schmidt Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Marie Friedrich Marie Friedrich Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Jacqueline Keye Jacqueline Keye Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Jiajia Wan Jiajia Wan The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China Search for more papers by this author Yue Qin Yue Qin orcid.org/0000-0001-9793-7964 National Center for Nanoscience and Technology, Beijing, China Search for more papers by this author Anja A Kühl Anja A Kühl iPATH.Berlin – Core Unit of the Charité, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Zhihai Qin Corresponding Author Zhihai Qin [email protected] The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China Search for more papers by this author Britta Siegmund Britta Siegmund Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Rainer Glauben Corresponding Author Rainer Glauben [email protected] orcid.org/0000-0003-2889-2525 Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Author Information Hao Wu1,2,3, Yijie Han4,5, Yasmina Rodriguez Sillke2,6, Hongzhang Deng7, Sophiya Siddiqui2,3, Christoph Treese2,8, Franziska Schmidt2,3, Marie Friedrich2,3, Jacqueline Keye2,3, Jiajia Wan1, Yue Qin9, Anja A Kühl10, Zhihai Qin *,1, Britta Siegmund2,‡ and Rainer Glauben *,2,‡ 1The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China 2Medical Department for Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 3Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany 4University of Chinese Academy of Sciences, Beijing, China 5Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 6Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany 7Department of Polymer Science and Engineering, Key Laboratory of Systems, Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China 8Berlin Institute of Health (BIH), Berlin, Germany 9National Center for Nanoscience and Technology, Beijing, China 10iPATH.Berlin – Core Unit of the Charité, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany ‡These authors contributed equally to this work ‡[Correction added on 13 November 2019, after first online publication: the affiliations have been corrected.] *Corresponding author. Tel: +86 371 6691 3632; E-mail: [email protected] *Corresponding author. Tel: +49 30 4505 14343; E-mail: [email protected] EMBO Mol Med (2019)11:e10698https://doi.org/10.15252/emmm.201910698 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Tumor-associated macrophages (TAMs) promote tumor growth and metastasis by suppressing tumor immune surveillance. Herein, we provide evidence that the immunosuppressive phenotype of TAMs is controlled by long-chain fatty acid metabolism, specifically unsaturated fatty acids, here exemplified by oleate. Consequently, en-route enriched lipid droplets were identified as essential organelles, which represent effective targets for chemical inhibitors to block in vitro polarization of TAMs and tumor growth in vivo. In line, analysis of human tumors revealed that myeloid cells infiltrating colon cancer but not gastric cancer tissue indeed accumulate lipid droplets. Mechanistically, our data indicate that oleate-induced polarization of myeloid cells depends on the mammalian target of the rapamycin pathway. Thus, our findings reveal an alternative therapeutic strategy by targeting the pro-tumoral myeloid cells on a metabolic level. Synopsis Tumor-associated macrophages (TAMs) are the main regulatory cell type in the tumor stroma as well as the microenvironment. This study describes how fatty acids polarize myeloid cells to TAMs and how this polarization is controlled by lipid droplet-dependent fatty acid metabolism. The fatty acid-enriched tumor environment itself was sufficient to induce the regulatory phenotype of TAMs, including the up-regulation of classical markers like CD206, IL-6, VEGFα, MMP9 or Arg1. The fatty acid-induced TAM polarization was lipid droplet dependent. mTORC2 activation played a critical role in the generation of the suppressive myeloid cell phenotype. Cell-specific inhibition of DGAT1 and 2 prevented oleate-induced polarization into immunosuppressive TAMs in vitro in murine and human cell culture systems as well as in vivo in a murine tumor model. Introduction Reprogramming of metabolic pathways guarantees the viability and proliferative capacity of cancer cells in a nutrient-poor environment (Pavlova & Thompson, 2016). These alterations include aerobic glycolysis (termed as Warburg effect; Warburg, 1956), increased glutamine uptake (Eagle, 1955), as well as amplified de novo fatty acid synthesis (Currie et al, 2013). Additionally, cancer cell-derived metabolites have been shown to contribute to the harsh tumor microenvironment (Anderson et al, 2017). In line, previous studies have already indicated decades ago that cancer cells but not stromal cells prefer to export fatty acids resulting in a fatty acid-enriched niche (Spector, 1967). Tumor-associated macrophages (TAMs) represent an abundant population in the multi-cellular tumor microenvironment. In fact, the infiltration and differentiation of TAMs correlate positively with all stages of tumor progression (Noy & Pollard, 2014). Characterized as M2-like macrophages, TAMs exert multiple pro-tumoral properties. TAMs not only hamper anti-tumor immune responses via regulating T and NK cell activation and apoptosis, but also facilitate angiogenesis and metastasis (Lin et al, 2007; Gajewski et al, 2013; Yeo et al, 2014). Accordingly, macrophage-targeting therapies led to positive results in murine tumor models. Clodronate-liposome-mediated abrogation of macrophages, for example, resulted in limited tumor progression in a murine xenograft model (Zeisberger et al, 2006). Furthermore, the inhibition of macrophage recruitment, for instance, via CCR2 silencing, was followed by a significant inhibition of tumor development in both solid and hematologic tumor models (Leuschner et al, 2011; Lesokhin et al, 2012). However, therapeutic strategies specifically targeting TAMs are still in early development stages (Cook & Hagemann, 2013). Previous studies identified a distinct metabolic pathway between pro- and anti-inflammatory macrophages. Activated pro-inflammatory macrophages rely on glycolysis to meet the rapid energy consumption, while alternatively activated macrophages prefer to use fatty acid oxidation (Biswas & Mantovani, 2012; Galván-Peña & O'Neill, 2014). Considering the fatty acid-enriched tumor microenvironment and the anti-inflammatory phenotype of TAMs, we propose a model in which extracellular fatty acids polarize the infiltrating monocytes into M2-like pro-tumoral macrophages. Here, we found that fatty acids, especially unsaturated fatty acids, polarize bone marrow-derived myeloid cells into an M2-like phenotype with a robust suppressive capacity. Lipid droplets (LDs) play an essential role in this process by regulating the catabolism of free fatty acids (FFA) for mitochondrial respiration. Mammalian target of the rapamycin (mTOR) inhibition eliminates LD-derived mitochondrial respiration and therefore immune suppression, indicating a regulatory role of the mTOR signaling pathway in this process. Furthermore, both intra-tumoral injection of LD-associated inhibitors and specific disruption of LD formation in myeloid cells via liposome-mediated delivery system attenuated tumor growth in an in vivo model. Finally, analysis of colon cancer patients confirmed the correlation between the accumulation of LDs in TAMs and the clinical stage of tumor. Our results provide a novel mechanism as well as a therapeutic target on myeloid cell differentiation and therefore on tumor escape from immune surveillance. Results Oleate-induced mitochondrial respiration regulates the suppressive phenotype of myeloid cells Previously, we identified a Gr1−CD11b+ subset within oleate-polarized myeloid cells with a potent T-cell suppressive capacity in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF; Wu et al, 2017). Here, we show that the Gr1−CD11b+ subset promotes tumor growth in vivo (Appendix Fig S1). To characterize this Gr1−CD11b+ population, fatty acid-treated myeloid cells were sorted and analyzed by microarray (Fig 1), which included further Gene Ontology (GO) analysis (Appendix Table S1). Interestingly, the expression level of differentially expressed genes in stearate-treated myeloid cells was close to the bovine serum albumin (BSA) control group, which differed significantly from the oleate-treated group, indicating a unique effect of oleate but not stearate on myeloid cell differentiation (Fig 1A). As expected, the core genes associated with de novo fatty acid synthesis and desaturation, for instance, Fasn as well as Fads2, Fads3 and Scd1 were, down-regulated (Fig 1B) when oleate was added as an external source. Remarkably, the LD formation-related genes, for instance, Dgat1 and Agpat9, were up-regulated. As GM-CSF could functionally polarize dendritic cells (DC) in vitro, the DC signature was analyzed first: the classical DC signature led to 13 out of 24 identified overlapping transcripts while indeed all of them were down-regulated in the oleate-treated group compared to the BSA control group (Fig 1B; Miller et al, 2012). Essential transcription factors including Flt3 and Btla that regulate the maturation of DCs were also down-regulated. Hence, we concluded that oleate potently suppresses GM-CSF-induced DC polarization on a transcriptional level. Former work from Herber et al (2010) revealed that LD containing DCs failed to present antigen. Indeed, 14 MHCII complex-associated genes including Ciita, the master regulator for MHCII expression, were down-regulated in the oleate group, compared with either BSA or stearate treatment (Fig 1B; GO term Go: 42613; Go: 2504), which was confirmed by flow cytometry (Fig 1C). In contrast to DCs, five out of 14 mature macrophage signature genes were differentially expressed, of which three were up-regulated (Gautier et al, 2012). From these data, we concluded that, following oleate treatment, the Gr1−CD11b+ population exhibits an immature macrophage phenotype. Flow cytometry staining of F4/80 and CD11b further confirmed this macrophage subset (Fig 1C). In addition, the innate immune response-associated genes, including Oas1a, Oas2, Oas3, Ifi202b, Irf7, and Tlr9, were all down-regulated, indicating a deficiency in anti-tumor immune response (Fuertes et al, 2013). Furthermore, a cluster of TAM-associated genes was up-regulated including the surface marker Mrc1, M2 phenotyping markers Arg1, Retnla, Chil3, as well as the functional markers Vegfa and Mmp9 (Fig 1B). An increased expression of the conventional TAM marker CD206+ as well as a robust arginase activity in the oleate-treated group, detected by flow cytometry, confirmed the mRNA expression analysis (Fig 1C). Recently identified surface markers which have been associated with the inhibitory phenotype of myeloid cells, including CD38 and CD73 (Beavis et al, 2012; Karakasheva et al, 2015), were also elevated in the oleate group as determined by microarray and flow cytometry (Fig 1C). Thus, these data indicate that during differentiation, oleate alone is sufficient to induce TAMs on a phenotypical and functional level. Figure 1. Oleate polarizes bone marrow-derived myeloid cells into immune suppressive tumor-associated macrophages A. Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1−CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group. B, C. Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor-associated macrophages (TAMs) phenotype, MHCII complex, and innate immune response are listed (B) and validated (C) via flow cytometry or catalytic activity assay. Data are expressed as mean ± SD from two to four independent experiments. Unpaired Student's two-tailed t-tests were performed to compare the expression level of indicated proteins in control and oleate groups. *P < 0.05; **P ≤ 0.01. Download figure Download PowerPoint Oleate-induced mitochondrial respiration regulates the suppressive phenotype of myeloid cells Prostaglandin E2 (PGE2) acts as an immune suppressive factor in malignancy niches (Torroella-Kouri et al, 2009). Our microarray data indicate that oleate treatment elevated the expression of ptgs1 (COX1), the main producer of PGE2 in eukaryotes. Thus, we were wondering whether COX1 contributes to the suppressive capacity of oleate-polarized myeloid cells. Myeloid cells were treated with celecoxib, an inhibitor of COX1, which effectively impaired the suppressive function of oleate-treated myeloid cells through diminishing nitric oxide (NO) production and arginase activity (Appendix Fig S2A and B). However, expression of CD38, CD206, and MHCII did not alter, indicating that PGE2 synthesis is downstream or independent of the oleate-induced differentiation cascade (Appendix Fig S2C). Fatty acid metabolism is required for membrane synthesis as well as for energy consumption during the polarization of myeloid cells. Indeed, purified Gr1−CD11b+ cells from the oleate-treated group revealed a significant increase of the mitochondrial respiratory capacity under both quiescent and stressed conditions (Fig 2A), as indicated by basal oxygen consumption, spare respiratory capacity, maximal respiration, adenosine triphosphate (ATP) production, and proton leak. In contrast, the basal level of glycolysis, as defined by extracellular acidification rate (ECAR), did not alter (Fig 2B). To determine the contribution of mitochondrial respiration in oleate-induced polarization of myeloid cells, the chemical inhibitor etomoxir was applied to block carnitine palmitoyltransferase 1 (CPT1), an enzyme associated with the outer mitochondrial membrane that transfers a long-chain acyl group from coenzyme A to carnitine, a process which is required to transport long-chain fatty acids into the mitochondrial matrix (Yao et al, 2018). Furthermore, it has been published that high concentrations of etomoxir (100 μM) directly impair mitochondrial respiration via decreasing the concentration of CoA in the cytosol or via inhibiting the mitochondrial respiratory complex I (Divakaruni et al, 2018; Yao et al, 2018). The treatment of etomoxir (40 μM) led to a reduction of mitochondrial respiration in both, the control and the oleate group (Fig 2C and D). Simultaneously, etomoxir exerted a comprehensive disruption of the oleate-induced effects, including the expression of CD206, CD38, and CD73 (Fig 2E and F), the inhibition of T-cell proliferation as well as NO production (Fig 2G–I), confirming the vital role of fatty acid oxidation in driving the maturation of CD206+ cells. David E. Sanin proved recently that PGE2 functions as a negative regulator of mitochondrial respiration in TAMs through modulating the malate–aspartate shuttle, thus explaining, why the reduction of PGE2 via COX1 inhibitor did not affect TAMs in our system (Sanin et al, 2018). Stearoyl-CoA desaturase-1 (SCD1) plays a crucial role in the endogenous production of unsaturated fatty acids. The SCD1 inhibitor CAY10566 served to estimate the contribution of endogenous unsaturated fatty acids on the polarization of CD206+ TAMs. Our data revealed that CAY10566 functionally impaired mitochondrial respiration and hampered the polarization of CD206+ myeloid cells in the control but not in the oleate group, indicating a compensatory effect of extracellular oleate (Appendix Fig S3). In summary, unsaturated fatty acid-derived mitochondrial respiration maintains the phenotype and function of TAMs in vitro. Figure 2. Oleate-induced mitochondrial respiration regulates the suppressive phenotype of myeloid cells A, B. Gr1−CD11b+ cells, polarized by the indicated treatment, were purified for mitochondrial respiration detection. The oxygen consumption rate (OCR) of these cells was monitored after the addition of oligomycin (OA; 1 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM), and the electron transport inhibitor rotenone and antimycin A (R/AA; 0.5 μM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production, and maximal respiration based on the OCR value were quantified. C, D. Forty micromolar etomoxir was added starting from day 0 of bone marrow polarization, followed by mitochondrial respiration assay by using the same amount of polarized Gr1−CD11b+ myeloid cells on day 7. The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified. E, F. Exemplary plots of the proportion of CD206+ cells, the mean fluorescence intensity (MFI) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry. G–I. T-cell proliferation assay was performed via co-culture with purified CD4+ T cells in the ratios of M (myeloid cells): T (T cells) = 1:30 (G, H). (I) Nitric oxide (NO) production from the co-culture supernatant was quantified by Griess reaction. Data information: Data are expressed as mean ± SD from two to four independent experiments and analyzed by either one-way analysis of variance (ANOVA) (B) or two-way ANOVA with Tukey's post hoc test (D, F, H, I). *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001. Source data are available online for this figure. Source Data for Figure 2 [emmm201910698-sup-0002-SDataFig2.xlsx] Download figure Download PowerPoint Lipid droplet-derived fatty acids facilitate mitochondrial respiration in myeloid cells Our previous data demonstrated an intimate correlation between the enrichment of LDs and the suppressive phenotype of MSC-2 cell line as well as primary myeloid cells (Wu et al, 2017). Thus, we proposed that LDs act as a stable source of fatty acids to maintain the regulatory phenotype of macrophages. Diacylglycerol O-acyltransferase (DGAT) is responsible for the import of FFAs into LDs, while a cascade of lipases including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MAGL) facilitate the depletion of LDs upon cell activation. Therefore, ATGL and HSL translocate to the LD membrane and cleave fatty acids from the stored triglycerides and therefore control the degradation of LDs. MAGL converts monoacylglycerols to the FFA and glycerol (Smirnova et al, 2006; Wang et al, 2009; Nomura et al, 2010; Fig 3A). We uncovered that inhibition of the catalytic activities of DGAT, ATGL, and MAGL attenuated oleate-induced mitochondrial respiration, especially ATP production (Fig 3B), as well as the expression of CD206 and their immune suppressive capacity (Fig 3C–F). These data indicate that in fact the export of FFAs from LDs controls the polarization of suppressive myeloid cells. Thus, we identified LDs as a novel source of fatty acids contributing to the polarization of TAMs. Figure 3. Lipid droplet-derived fatty acids facilitate mitochondrial respiration in myeloid cells A. The formation and utilization of lipid droplets in eukaryotes. Five micromolar combination of DGAT inhibitors (DGAT1 inhibitor A922500 and DGAT2 inhibitor PF-06424439), 40 μM ATGL inhibitor atglistatin (Atg), or 5 μM MAGL inhibitor MJN110 was added to the bone marrow polarization system in the presence of 40 ng/ml GM-CSF and indicated compounds for 7 days. B. The oxygen consumption rate (OCR) of 1 × 105 purified Gr1−CD11b+ cells was monitored as described in Fig 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production, and maximal respiration based on the OCR value were quantified. C. The polarization state was evaluated via the expression of CD206. D. T-cell proliferation assay was performed employing co-culture with purified CD4+ T cell in the variant ratios. E. The lipid droplet accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index was calculated. F. Nitric oxide (NO) concentration in the co" @default.
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