{ "title": "Sphingosine d18:1 promotes nonalcoholic steatohepatitis by inhibiting macrophage HIF-2\u03b1", "pre_title": "Sphingosine d18:1 Promotes Nonalcoholic Steatohepatitis by Inhibiting Macrophage HIF-2\u03b1", "journal": "Nature Communications", "published": "04 June 2024", "supplementary_0": [ { "label": "Supplementary Information", "link": "https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_MOESM1_ESM.pdf" }, { "label": "Peer Review File", "link": "https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_MOESM2_ESM.pdf" }, { "label": "Reporting Summary", "link": "https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_MOESM3_ESM.pdf" } ], "supplementary_1": [ { "label": "Source Data", "link": "https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_MOESM4_ESM.zip" } ], "supplementary_2": NaN, "source_data": [ "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262135", "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166504", "/articles/s41467-024-48954-2#Sec30" ], "code": [], "subject": [ "Hepatocytes", "Inflammation", "Non-alcoholic fatty liver disease", "Non-alcoholic steatohepatitis" ], "license": "http://creativecommons.org/licenses/by/4.0/", "preprint_pdf": "https://www.researchsquare.com/article/rs-3092076/v1.pdf?c=1717585607000", "research_square_link": "https://www.researchsquare.com//article/rs-3092076/v1", "nature_pdf": "https://www.nature.com/articles/s41467-024-48954-2.pdf", "preprint_posted": "10 Jul, 2023", "research_square_content": [ { "section_name": "Abstract", "section_text": "Non-alcoholic steatohepatitis (NASH) is a severe type of the non-alcoholic fatty liver disease (NAFLD). NASH is a growing global health concern due to its increasing morbidity, lack of well-defined biomarkers and lack of clinically effective treatments. Using metabolomic analysis, the most significantly changed active lipid sphingosine d18:1 [So(d18:1)] was selected from NASH patients. So(d18:1) inhibits macrophage HIF-2\u03b1 as a direct inhibitor and promotes the activation of NLRP3 inflammasome. Macrophage-specific HIF-2\u03b1 knockout and overexpression mice verified the effect of HIF-2\u03b1 on NASH progression. Importantly, the HIF-2\u03b1 stabilizer FG-4592 alleviated liver inflammation and fibrosis in NASH, which indicated that macrophage HIF-2\u03b1 was a potential drug target for NASH treatment. Overall, this study confirms that So(d18:1) promotes NASH and clarifies that So(d18:1) inhibits the transcriptional activity of HIF-2\u03b1 in liver macrophages by suppressing the interaction of HIF-2\u03b1 with ARNT, suggesting that macrophage HIF-2\u03b1 may be a new target for the treatment of NASH.Health sciences/Gastroenterology/Hepatology/Liver diseases/Non-alcoholic steatohepatitisHealth sciences/Gastroenterology/Hepatology/Liver diseases/Non-alcoholic fatty liver diseaseNASHmacrophageHIF-2\u03b1sphingosine", "section_image": [] }, { "section_name": "Additional Declarations", "section_text": "There is NO Competing Interest.", "section_image": [] }, { "section_name": "Supplementary Files", "section_text": "Supplementary.pdf", "section_image": [] } ], "nature_content": [ { "section_name": "Abstract", "section_text": "Non-alcoholic steatohepatitis (NASH) is a severe type of the non-alcoholic fatty liver disease (NAFLD). NASH is a growing global health concern due to its increasing morbidity, lack of well-defined biomarkers and lack of clinically effective treatments. Using metabolomic analysis, the most significantly changed active lipid sphingosine d18:1 [So(d18:1)] is selected from NASH patients. So(d18:1) inhibits macrophage HIF-2\u03b1 as a direct inhibitor and promotes the inflammatory factors secretion. Male macrophage-specific HIF-2\u03b1 knockout and overexpression mice verified the protective effect of HIF-2\u03b1 on NASH progression. Importantly, the HIF-2\u03b1 stabilizer FG-4592 alleviates liver inflammation and fibrosis in NASH, which indicated that macrophage HIF-2\u03b1 is a potential drug target for NASH treatment. Overall, this study confirms that So(d18:1) promotes NASH and clarifies that So(d18:1) inhibits the transcriptional activity of HIF-2\u03b1 in liver macrophages by suppressing the interaction of HIF-2\u03b1 with ARNT, suggesting that macrophage HIF-2\u03b1 may be a potential target for the treatment of NASH.", "section_image": [] }, { "section_name": "Introduction", "section_text": "With lifestyle changes, nonalcoholic fatty liver disease (NAFLD) has become a major chronic disease in contemporary society1. NAFLD is a chronic metabolic disease characterized by excessive accumulation of fat in hepatocytes. NAFLD can be divided into simple fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH)2. Chronic liver injury in NASH significantly increases the risk of end-stage liver diseases (such as cirrhosis and liver cancer). However, there is no effective drug for NASH in clinical practice3. Therefore, clarifying the key molecular mechanism of the occurrence and development of NASH will contribute to developing new strategies for anti-NASH treatment.\n\nThe classical theory of NASH pathogenesis is that NASH is caused by the excessive accumulation of lipids in hepatocytes. Then, extreme oxidative stress and inflammation further induce hepatocyte death and the development of inflammation and fibrosis4. Sphingolipids are lipids with high biological activity and are one of the main factors affecting the progression of NASH. Sphingolipids mainly include ceramide, sphingosine-1-phosphate (S1P), sphingomyelins, and sphingosine5. Previous studies have found changes in ceramide and S1P levels in NASH patients6. However, they both failed to act as sensitive biomarkers to guide disease diagnosis in NASH because of their widespread variation in many early-stage NAFLD patients. Sphingosine has not only been found to vary in NAFL7,8 but has even been found to be useful as a biomarker to predict cirrhosis9.\n\nHere, we found that So(d18:1) increases significantly in patients with NASH by metabolomics profiling analysis. So(d18:1) promotes liver inflammation and fibrosis in the NASH model. RNA-seq data revealed that So(d18:1) inhibits HIF-2\u03b1 expression. Macrophage-specific knockout or overexpression of HIF-2\u03b1 has been used to clarify the role of macrophage HIF-2\u03b1 in NASH development. Mechanistically, So(d18:1) inhibits macrophage HIF-2\u03b1 by inhibiting its combination with ARNT and then promotes the secretion of inflammatory factors. Notably, we found that the pharmacological activation of macrophage HIF-2\u03b1 by FG-4592, a HIF prolyl hydroxylase inhibitor that is approved for the treatment of anaemia in China, had preventive effects on NASH in mice. This work suggests that macrophage HIF-2\u03b1 is a potential target for the treatment of NASH.", "section_image": [] }, { "section_name": "Results", "section_text": "In the Chinese patient population, we employed a metabolomics screen of NASH patients and healthy volunteers (Supplementary Table\u00a01). The results showed that the changes in the sphingolipid pathway are the most concentrated, significant and dramatic compared to other lipids that are considered to change routinely (Fig.\u00a01A). After that we further examined the whole sphingolipidome using targeted metabolomics (Fig.\u00a01B). Principal component analysis (PCA) showed a clear separation between the healthy volunteers and NASH patients (Fig.\u00a01C). The VIP score indicated a significant increase in the levels of several sphingolipids, especially So(d18:1) (Fig.\u00a01D).\n\nMetabolic analysis of serum samples collected from NASH patients (n\u2009=\u200916) and healthy control (n\u2009=\u200916). A Clustering heatmap of the metabolic pathway, colours represent the intensity of response. B Targeted metabonomic detection of sphingolipids, colours represent the intensity of response. C PLS-DA analysis of sphingolipids in serum of patients. D VIP score plot of the difference sphingolipids between the two groups. E, F Serum So(d18:1) concentration in healthy volunteers (n\u2009=\u200916 biologically independent samples) and NASH patients (n\u2009=\u200915 biologically independent samples) (E) and different stages of NASH patients classified by NAS scores, NAS-3 (n\u2009=\u20096 biologically independent samples), NAS-4 (n\u2009=\u20096 biologically independent samples), NAS-5 (n\u2009=\u20093 biologically independent samples) (F). G Serum concentration of So(d18:1) in different time points of NASH modelling mice (n\u2009=\u20096 biologically independent animals). H\u2013J Correlative analysis of So(d18:1) concentration in serum with ALT (Alanine aminotransferase) (H), AST (Aspartate aminotransferase) (I) and Fibroscan index (J). Correlations between variables were assessed by linear regression analysis. Linear correction index R square and P-values were calculated. Data are the means\u2009\u00b1\u2009s.e.m. Statistical analysis was performed using two-tailed Student\u2019s t-tests (E), One-way ANOVA with Dunnett\u2019s T3 post hoc test (F) and Kruskal\u2013Wallis test with Dunn\u2019s test (G).\n\nIn the human cohort, So(d18:1) accumulated largely in the serum of NASH patients (Fig.\u00a01E, F). Moreover, the concentration of So(d18:1) was positively correlated with serum ALT, AST levels and Fibrosacn index (Fig.\u00a01H\u2013J). To further validate this phenotype, we also fed the mice with CDAA-HFD for 8 weeks to establish the NASH mice model. Serum So(d18:1) concentrations were assayed in NASH model mice, and the trend of increasing serum So(d18:1) concentrations in mice was exactly the same as the trend of increasing ALT and AST levels (Fig.\u00a01G, Fig.\u00a0S1A, B). These results suggested that So(d18:1) concentrations may be closely related to NASH progression. However, So(d18:1) relative concentration in whole liver tissue didn\u2019t increase with the development of NASH (Fig.\u00a0S1C). The origin of So(d18:1) may need further investigation.\n\nIn our sphingolipidome results, the upstream and downstream metabolites of sphingosine, ceramide and S1P, were also altered in content. In our previous study, we found that ceramide was enriched in NASH patients similarly6. But ceramides did not increase more with disease progression (Fig.\u00a0S1D). S1P and the other types of sphingosines also didn\u2019t show any growth trends during the progression of NASH (Fig.\u00a0S1E\u2013I). These results further demonstrated the unique indicative role of So(d18:1) in the progression of NASH.\n\nHepatic steatosis and lobular inflammation are two important features of NASH. We analysed the changes of So(d18:1) levels in these two aspects. There was no significant increase in the So(d18:1) level as hepatic steatosis progressed (Fig.\u00a0S1J). However, the So(d18:1) concentration shows a gradual trend of increase with the aggravation of lobular inflammation (Fig.\u00a0S1K), which suggests that the function of So(d18:1) may be related to lobular inflammation.\n\nTo test whether So(d18:1) is involved in the progression of NASH, CDAA-HFD-fed mice were intraperitoneally injected with vehicle, So(d16:1) or So(d18:1), respectively. So(d16:1) was used as a control to show the specific effects of So(d18:1). Detection of the concentration of So(d18:1) in the plasma after injection shows that the growth of So(d18:1) is similar to the concentration in the plasma of NASH patients, which can mimic the function of So(d18:1) in the pathological state (Fig.\u00a0S2A).\n\nLiver weight and the ratio of liver weight to body weight were significantly increased in mice injected with So(d18:1) compared with mice injected with vehicle\u00a0(Figs.\u00a02A, B and S2B). The levels of ALT and AST in the serum of mice injected with So(d18:1) were significantly higher than those in control mice (Fig.\u00a02C, D), which suggests that So(d18:1) exacerbated liver damage in mice. While there were no differences in liver triglyceride (TG), serum TG and serum non-esterified fatty acid (NEFA) levels, there was also no difference in liver and serum cholesterol (CE) levels (Fig.\u00a0S2C\u2013G). For a clearer image of the liver damage in mice, we made pathological sections and performed H&E staining and Sirius red staining. The pathological sections showed that So(d18:1) treatment increased the fibrosis, lobular inflammation and the histology score of NAFLD activity but did not affect the histology score of hepatic steatosis (Fig.\u00a02E\u2013J). Consistently, the mRNA expression of inflammation genes and fibrosis genes were significantly upregulated in the liver of the So(d18:1) group compared with that of the vehicle group (Fig.\u00a02K, L), while the lipid metabolism genes were mostly not different (Fig.\u00a0S2H). The results showed that the administration of So(d16:1) to mice of the NASH model did not further exacerbate the disease phenotype of NASH (Figs.\u00a02 and\u00a0S2). This reinforced the significant role of So(d18:1). Collectively, these results suggest that So(d18:1) can exacerbate lobular inflammation and fibrosis in the livers of NASH mice.\n\nCDAA-HFD-fed mice were treated with vehicle, sphingosine 16:1 or sphingosine 18:1 for 8 weeks (n\u2009=\u20096 mice/group). A Liver weights. B Ratios of liver mass to body mass. C Serum ALT. D Serum AST. E Representative H&E (up), and Sirius Red (down) staining of liver sections. The circles marked the inflammation foci. n\u2009=\u20093 mice per group, 3 images per mouse. Scale bar is 100\u2009\u03bcm. F The percentage of fibrosis area. G\u2013J Histology scores of hepatic steatosis (G), lobular inflammation (H), ballooning (I), and NAFLD activity (J). K, L Relative mRNA levels of genes related to hepatic inflammation (K) and fibrosis (L). n\u2009=\u20096 biologically independent samples. Data are the means\u2009\u00b1\u2009s.e.m. A, C, D, F, K, L Statistical analysis was performed using One-way ANOVA; B, G\u2013J Col5a2 in L, statistical analysis was performed using Kruskal\u2013Wallis test with Dunn\u2019s test.\n\nSo(d18:1) can exacerbate lobular inflammation in the liver of NASH, suggesting that it mainly alters the immune status of the liver, so we focused on immune cells for an in-depth study. To confirm the changes of various immune cells during the development of NASH, a set of public single-cell RNA-sequencing data from the livers of NASH mice were located and analysed10. The results showed an increase in all kinds of immune cells in the livers of NASH mice. However, the largest proportion of these cells were macrophages and monocytes. Importantly, they were recruited to the livers much earlier than other immune cells (Fig.\u00a0S3A, B). We therefore wanted to see whether So(d18:1) would also cause changes in macrophage proportion. We administered So(d18:1) or So(d16:1) intraperitoneally to chow-diet fed mice for 1 week. Results showed that only So(d18:1) increased the proportion of liver macrophages among all immune cells (Figs. S3C and 3A, B).\n\nA, B Flow cytometric and statistical analysis of chow-diet fed mice treated with control (5% CMC-Na), So(d16:1) and So(d18:1) (n\u2009=\u20095 per group). C GO:BP pathway enrichment showing the transcriptional level changes of some immune-related pathways. (n\u2009=\u20094 biologically independent samples). D Epas1 targets enrichment. E Relative mRNA levels of Hif2\u03b1 and its downstream target genes in macrophages treated with vehicle, So(d18:1), So(d16:1), So(d20:1) or Sa(d18:1). (n\u2009=\u20093). F Representative immunoblot analysis of HIF-2\u03b1 of BMDMs isolated from wild-type mice stimulated with the vehicle and So(d18:1) and BMDMs isolated from LysMHif2\u03b1LSL/LSL and Hif2\u03b1\u25b3LysM BMDMs stimulated with the vehicle.\u00a0This experiment was repeated 3 times independently with similar results. G Representative immunoblot analysis of pro-caspase-1 and caspase-1 from Hif2\u03b1+/+ and LysMHif2\u03b1LSL/LSL BMDMs that were treated with So(d18:1) or not under NLRP3 inflammasome stimulation. This experiment was repeated 3 times independently with similar results. H, I Protein level of IL-1\u03b2 (H), IL-18 (I) from Hif2\u03b1+/+ and LysMHif2\u03b1LSL/LSL BMDMs treated with So(d18:1) or not under NLRP3 inflammasome stimulation. (n\u2009=\u20096 biologically independent samples). Data are the means\u2009\u00b1\u2009s.e.m. B, E, H, I Statistical analysis was performed using One-Way ANOVA test; Spint in (E), statistical analysis was performed using the Kruskal\u2013Wallis test. C One-sided hypergeometric distribution test, using Benjamin Hochberg method for p-value correction. D One-sided permutation test without p-value correction.\n\nTo search for the mechanisms by which So(d18:1) promotes macrophage activation, we treated mouse bone marrow-derived macrophages (BMDM) with So(d18:1) or control vehicle under inflammatory stimulation and performed RNA sequencing to explore the changed gene pathways. GO:BP pathway enrichment showed that hypoxia-related pathways were changed significantly between the control and So(d18:1) groups (Fig.\u00a03C). There are two transcription factors that play a major role in the hypoxia-related signalling pathway, HIF-1\u03b1 and HIF-2\u03b1. We further targeted the signalling pathways regulated by these two transcription factors for enrichment analysis. The GSEA enrichment analysis revealed transcriptional changes in the HIF-2\u03b1-regulated signalling pathway (Fig.\u00a03D), while the HIF-1\u03b1 signalling pathway was not changed (Fig.\u00a0S3D). Flow cytometric analysis could also show that CDAA-HFD feeding suppresses HIF-2\u03b1 expression in liver macrophages (Fig.\u00a0S3F).\n\nTo validate the RNA-seq results, we treated mouse BMDMs with So(d18:1) and other sphingolipids as controls. The results showed that the transcription levels of the Hif2\u03b1 gene were not changed, but its downstream gene Vegf decreased after So(d18:1) treatment (Fig.\u00a03E). We also detected Hif1\u03b1 and its downstream genes and their expression levels were unchanged (Fig.\u00a0S3E). As for the protein levels of HIF-2\u03b1, the results showed that So(d18:1) treatment could significantly inhibit the protein expression of HIF-2\u03b1 (Fig.\u00a03F). In addition to the in vitro experiments, we also detected the expression of HIF-2\u03b1 in liver macrophages from CDAA-HFD diet fed mice treated with vehicle (5% CMC-Na) or So(d18:1) using flow cytometry, and the results showed that So(d18:1) significantly inhibited the expression of HIF-2\u03b1 in liver macrophages (Fig.\u00a0S3G).\n\nIntrahepatic macrophages IL-1\u03b2 and IL-18 secretion due to NLRP3 inflammasome activation is an important mechanism that promotes the progression of NASH11. Our previous study also found that macrophage HIF-2\u03b1 could suppress NLRP3 inflammasome activation by inhibiting CPT1A12. Consistently, western blot\u00a0analysis showed that So(d18:1) administration increased cleaved-caspase-1, indicating So(d18:1) could increase inflammasome assembly therefore increase Caspase-1 cleavage, while HIF-2\u03b1 overexpression could quell the stimulation caused by So(d18:1) (Fig.\u00a03G). IL-1\u03b2 and IL-18 secretion levels also confirmed that only So(d18:1) promoted inflammasome activation (Fig.\u00a0S3H, I), but not in HIF-2\u03b1 overexpressing macrophages (Fig.\u00a03H, I). Besides, So(d18:1) didn\u2019t damage the hepatocytes or influence the fibrosis-related genes in hepatic stellate cells directly (Fig. S3J, K).\n\nThese results indicate that So(d18:1) promotes the inflammatory factors secretion of macrophages through inhibition of HIF-2\u03b1, which may be the cellular mechanism by which So(d18:1) promotes hepatic inflammation in NASH.\n\nTo investigate whether HIF-2\u03b1 in macrophages can influence NASH disease progression, we fed Hif2\u03b1fl/fl and Hif2\u03b1\u0394Lysm mice a GAN diet (40% fat-powered, 20% fructose, and 2% cholesterol) for 24 weeks to compare the severity of inflammation and fibrosis in the liver. There was no significant difference in body weight between the two groups of mice (Fig.\u00a0S4A). Liver weight and the ratio of liver weight to body weight were significantly increased in Hif2\u03b1\u0394Lysm mice compared with Hif2\u03b1fl/fl mice (Fig.\u00a04A, B). Moreover, the levels of ALT and AST in the serum of Hif2\u03b1\u0394Lysm mice were significantly higher than those in Hif2\u03b1fl/fl mice, suggesting that knockdown of Hif2a exacerbates the disease symptoms of NASH (Fig.\u00a04C, D). Next, we examined the changes in lipids in the liver tissue and plasma of the two groups of mice. The results revealed that the concentrations of serum TG, CE, and NEFAs and hepatic TG and CE were not significantly different between Hif2\u03b1fl/fl mice and Hif2\u03b1\u0394Lysm mice (Fig.\u00a0S4C\u2013F). This result suggests that the knockdown of macrophage Hif2a does not affect total lipid metabolism or consequently exacerbate lipid accumulation in the liver.\n\nEight-week-old male Hif2\u03b1fl/fl and Hif2\u03b1\u0394Lysm mice were administered a GAN diet for 24 weeks (SPF, n\u2009=\u20096 mice/group). A Liver weights. B Ratios of liver mass to body mass. C Serum ALT. D Serum AST. E Representative H&E (up), and Sirius Red (down) staining of liver sections. The circles marked the inflammation foci. n\u2009=\u20093 mice per group, 3 images per mouse. Scale bar is 200\u2009\u03bcm. F The percentage of fibrosis area. G\u2013J Histology scores of hepatic steatosis (G), lobular inflammation (H), ballooning (I), and NAFLD activity (J). K, L Relative mRNA levels of genes related to hepatic inflammation (K) and fibrosis (L). n\u2009=\u20096 biologically independent samples. Data are the means\u2009\u00b1\u2009s.e.m. A\u2013D, F, K, L Statistical analysis was performed using two-tailed Student\u2019s t-tests; G\u2013J Col2a1 in (L), statistical analysis was performed using two-tailed Mann\u2013Whitney U-tests.\n\nTo further determine the changes in the levels of inflammation and fibrosis within the mouse liver to determine the progression of NASH, pathological sections were made from the livers of the two groups of mice to observe the extent of liver injury in the mice (Fig.\u00a04E). The degree of hepatic steatosis was consistent between the two groups of mice (Fig.\u00a04G), and there was no significant difference in the ballooning score (Fig.\u00a04\u0399). However, mice in the Hif2\u03b1\u0394Lysm group had more foci of inflammation in the liver, with a large number of mononuclear macrophages diffusely distributed and a significantly higher inflammation score in the liver lobules than in the Hif2\u03b1fl/fl group (Fig.\u00a04\u0397). The sections were also stained with Sirius red (Fig.\u00a04\u0395), and the fibrosis area was quantified to show that the Hif2\u03b1\u0394Lysm group had a significantly greater fibrosis area than that of the Hif2\u03b1fl/fl group (Fig.\u00a04F). These results demonstrate that macrophage Hif2a knockdown can indeed significantly exacerbate NASH symptoms and promote inflammatory activation and fibrosis formation.\n\nConsistently, the mRNA expression of inflammation genes and fibrosis genes was significantly upregulated in the livers of Hif2\u03b1\u0394Lysm mice compared with that of Hif2\u03b1fl/fl mice (Fig.\u00a04K, L), while the lipid metabolism genes were not different between the two groups (Fig.\u00a0S4G). Collectively, these data showed that genetic disruption of macrophage-specific HIF-2\u03b1 accelerated hepatic inflammation and fibrosis but did not affect hepatic steatosis.\n\nTo further verify the role of macrophage HIF-2\u03b1 overexpression in NASH and investigate whether macrophage-specific HIF-2\u03b1 overexpression could confront the effects of So(d18:1) on NASH in vivo, Hif2\u03b1+/+ and LysMHif2\u03b1LSL/LSL mice were fed a CDAA-HFD with vehicle or So(d18:1) injection for 8 weeks. There was no significant difference in body weight (Fig.\u00a0S5A). The liver weight and the ratio of liver weight to body weight decreased in LysMHif2\u03b1LSL/LSL mice compared with Hif2\u03b1+/+ mice (Fig.\u00a05A, B).\n\nEight-week-old male Hif2\u03b1+/+ and LysMHif2\u03b1LSL/LSL mice treated with or without So(d18:1) by daily intraperitoneal injection under CDAA-HFD for 8 weeks (SPF, n\u2009=\u20096 mice/group). A Liver weights. B Ratios of liver mass to body mass. C Serum ALT. D Serum AST. E Representative H&E (up), and Sirius Red (down) staining of liver sections. The circles marked the inflammation foci. n\u2009=\u20093 mice per group, 3 images per mouse. Scale bar is 100\u2009\u03bcm. F The percentage of fibrosis area. G\u2013J Histology scores of hepatic steatosis (G), lobular inflammation (H), ballooning (I), and NAFLD activity (J). K Relative mRNA levels of genes related to hepatic inflammation. L Relative mRNA levels of genes related to hepatic fibrosis. n\u2009=\u20096 biologically independent samples. Data are the means\u2009\u00b1\u2009s.e.m. A\u2013D, F, K, L Statistical analysis was performed using one-way ANOVA; G\u2013J Ccl2 in (K), Timp1, Col3a1, Col4a2 and Col5a2 in (L), statistical analysis was performed using the Kruskal\u2013Wallis.\n\nThe levels of ALT and AST in the serum were significantly lower in LysMHif2\u03b1LSL/LSL mice than in Hif2\u03b1+/+ mice (Fig.\u00a05C, D), suggesting that Hif2a overexpression can protect the liver and reduce liver injury. Moreover, macrophage-specific HIF-2\u03b1 overexpression lowered the ALT and AST lever in LysMHif2\u03b1LSL/LSL\u2009+\u2009So(d18:1) group compared with Hif2\u03b1+/+ + So(d18:1) group (Fig.\u00a05C, D). We also measured TG, total CE and NEFA levels in the liver and plasma to investigate whether macrophage-specific Hif2a overexpression could reduce fat accumulation in the liver, but there were no differences in any of these parameters (Fig.\u00a0S5B\u2013F).\n\nThe liver tissues of the four groups of mice were also paraffin sectioned and stained with H&E and Sirius red. The H&E staining results showed that there was no significant difference in the steatosis scores and hepatocyte ballooning scores (Fig.\u00a05E, G, I). However, the LysMHif2\u03b1LSL/LSL group mice had fewer inflammatory foci in the liver, so their hepatic lobular inflammation scores were significantly lower than those of the Hif2\u03b1+/+ group mice (Fig.\u00a05H) and the final calculated NAS of the LysMHif2\u03b1LSL/LSL group mice was significantly lower than that of the Hif2\u03b1+/+ group mice (Fig.\u00a05J). We next examined Sirius red-stained sections, and it was evident that intrahepatic fibrosis production was reduced in the LysMHif2\u03b1LSL/LSL group of mice (Fig.\u00a05E, F). So(d18:1) significantly increased the lobular inflammation, the calculated NAS and the fibrosis area in Hif2\u03b1+/+ mice, but not in LysMHif2\u03b1LSL/LSL mice (Fig.\u00a05E\u2013J). Importantly, the lobular inflammation scores, NAS and fibrosis area increased by So(d18:1) treatment were alleviated by the overexpression of HIF2a as comparing LysMHif2\u03b1LSL/LSL\u2009+\u2009So(d18:1) group with Hif2\u03b1+/+ + So(d18:1) group (Fig.\u00a05E\u2013J).\n\nConsistently, the mRNA expression of inflammation-related genes and fibrosis-related genes was significantly downregulated in the livers of LysMHif2\u03b1LSL/LSL mice compared with Hif2\u03b1+/+ mice (Fig.\u00a05K, L), while the lipid metabolism-related genes were not different (Fig.\u00a0S5G). So(d18:1) increased the mRNA level of inflammation-related genes and fibrosis in Hif2\u03b1+/+ mice, but not in LysMHif2\u03b1LSL/LSL mice (Fig.\u00a05K, L). Moreover, the mRNA expression of inflammation-related genes and fibrosis-related genes were reduced by the overexpression of HIF2a as comparing LysMHif2\u03b1LSL/LSL\u2009+\u2009So(d18:1) group with Hif2\u03b1+/+\u2009+\u2009So(d18:1) group (Fig.\u00a05K, L). Collectively, these data showed that macrophage-specific HIF-2\u03b1 overexpression ameliorated hepatic inflammation and fibrosis but did not affect hepatic steatosis. Moreover, macrophage-specific HIF-2\u03b1 overexpression confronted the effects of So(d18:1) on NASH in vivo.\n\nIn previous results, we have verified that So(d18:1) could promote NLRP3 inflammasome activation in macrophages and identified HIF-2\u03b1 as a key transcription factor by which So(d18:1) alters the inflammatory state of macrophages. Therefore, how does the increased So(d18:1) in NASH patients affect HIF-2\u03b1 protein function in macrophages? We conducted a more in-depth mechanistic study to address this question. To determine whether So(d18:1) could inhibit HIF-2\u03b1 transcriptional activity, we constructed a HIF response element (HRE)-based luciferase reporter assay. Fluorescein detection showed that So(d18:1) could significantly inhibit the transcriptional activity of HIF-2\u03b1 (Fig.\u00a06A). But So(d18:1) couldn\u2019t inhibit HIF-1\u03b1 transcription (Fig.\u00a06B). The transcriptional action of HIF-2\u03b1 requires binding to the ARNT subunit. Thus, we utilized a mammalian two-hybrid system that could further verify that So(d18:1) repressed the transcriptional function of HIF-2\u03b1 by inhibiting the binding of ARNT (Fig.\u00a06C). In addition, co-immunoprecipitation revealed that So(d18:1) disrupts the binding of HIF-2\u03b1 to ARNT (Fig.\u00a06D), but doesn\u2019t disrupt the direct binding of HIF-1\u03b1 to ARNT (Fig.\u00a06E).\n\nA HRE-based luciferase assay in HEK293T transfected with HIF-2\u03b1, followed by treatment of PT2385, So(d18:1), So(d16:1), So(d20:1), Sa(d18:1) (n\u2009=\u20093 biologically independent samples). B HRE-based luciferase assay in HEK293T transfected with HIF-1\u03b1, followed by treatment of PX-478, So(d18:1) (n\u2009=\u20096 biologically independent samples). C Schematic and experimental representation of PT2385 or So(d18:1) mediated HIF-2\u03b1-ARNT interaction by pG5GAL4 luciferase assay followed by pBIND-HIF-2\u03b1 and pACT-ARNT transfection of HEK293T cells (n\u2009=\u20095). D Co-immunoprecipitation for ARNT and HIF-2\u03b1 in HEK293T cells transfected with ARNT and the oxygen-stable HIF-2\u03b1 triple mutant (HIF-2\u03b1\u2122) plasmid treated with control solvent, So(d18:1) or PT2385, PT2385 and So(d18:1) could inhibit HIF-2\u03b1 to bind to ARNT. This experiment was repeated 3 times independently with similar results. E Co-immunoprecipitation for ARNT and HIF-1\u03b1 in HEK293T cells transfected with ARNT and the oxygen-stable HIF-2\u03b1 triple mutant (HIF-2\u03b1\u2122) plasmid treated with So(d18:1) (20\u2009\u03bcM) or TAY-cyclo (20\u2009\u03bcM). This experiment was repeated 3 times independently with similar results. F Molecule docking prediction of So(d18:1) binding sites in HIF-2\u03b1 PAS-B domain. G schematic diagram of site missense mutation experiment. PT2385 and So(d18:1) could inhibit normal HIF-2\u03b1 transcription ability but not HIF-2\u03b1 with missense mutations. (n\u2009=\u20096 biologically independent samples). H Cpt1a promoter rHRE constructs plasmid were co-transfected with HIF-2\u03b1\u2122 followed by control solvent or So(d18:1) treatment. So(d18:1) could inhibit HIF-2\u03b1 binding ability to rHRE. (n\u2009=\u20093 biologically independent samples). Data are the means\u2009\u00b1\u2009s.e.m. A\u2013C, G (WT), statistical analysis was performed using One-way ANOVA. G(mutant), statistical analysis was performed using Kruskal\u2013Wallis test with Dunn\u2019s test. H Statistical analysis was performed using two-tailed Student\u2019s t-tests.\n\nHIF-2\u03b1 has a hydrophobic pocket PAS-B domain to bind with ARNT13. Structural prediction by docking revealed some potential for So(d18:1) to fill into this hydrophobic pocket (Fig.\u00a06F). We then constructed a HIF-2\u03b1 plasmid with two proven missense mutations (S304M and G323E) in the pocket which disabled other molecules to bind with HIF-2\u03b114. Luciferase reporter system was performed, and the results showed that So(d18:1) could normally inhibit the binding of wild-type HIF-2\u03b1 to ARNT but not that of mutant HIF-2\u03b1 to ARNT (Fig.\u00a06G). From these results, we learned that So(d18:1) may fill into the hydrophobic pocket of HIF-2\u03b1 and thereby inhibit the binding of HIF-2\u03b1 to ARNT, which impedes HIF-2\u03b1 entry into the nucleus for transcriptional regulation. HIF-2\u03b1 that remains in the cytoplasm is very easily hydrolysed and therefore protein levels are reduced. This finding also explained why So(d18:1) can only change the protein expression level of HIF-2\u03b1 but not the mRNA expression level.\n\nHIF-2\u03b1 regulates metabolism reprogramming and NLRP3 inflammasome activation by binding to the rHRE region on the Cpt1a promoter12. We therefore transfected a luciferase reporter gene plasmid containing a Cpt1a rHRE region with a HIF-2\u03b1 plasmid or empty plasmid into cells, treated with control solvent or So(d18:1) and observed the fluorescence activity ratio. The results showed that in the vehicle group, the fluorescence values of HIF-2\u03b1TM-transfected cells were lower than those with empty plasmid, indicating that overexpression of HIF-2\u03b1 inhibited the transcription of Cpt1a rHRE-linked luciferase. In contrast, the overexpression of HIF-2\u03b1 in the So(d18:1) group did not affect the transcription of Cpt1a rHRE-linked luciferase, as it was unable to bind to ARNT and localize to the rHRE region in the nucleus, so the fluorescence values of HIF-2\u03b1TM plasmid-transfected cells were similar to the fluorescence values of the empty plasmid group (Fig.\u00a06H).\n\nThe above results suggest that So(d18:1) could inhibit the binding of HIF-2\u03b1 with ARNT to inhibit the transcriptional activity of HIF-2\u03b1, thus influencing the expression of downstream genes regulated by HIF-2\u03b1. These results also suggest the possibility that lipids bind directly to transcription factors and regulate their functions, showing us an interesting mechanism by which lipids influence cellular metabolism.\n\nWe further investigated the therapeutic effect of the specific HIF-2\u03b1 agonist FG-4592 in treating NASH. SPF mice were given a CDAA-HFD diet for 8 weeks and were administered vehicle or FG-4592 (25\u2009mg/kg) by intraperitoneal injection. At the end of the treatment, there was no significant difference in body weight between the two groups of mice (Fig.\u00a0S6A), but there was a significant reduction in liver weight (Fig.\u00a07A), as well as a significant reduction in the calculated liver weight/body weight ratio (Fig.\u00a07B). Measurement of the blood levels of ALT and AST showed significant decreases in both transaminase levels suggestive of liver injury (Fig.\u00a07C, D). To assess whether FG-4592 could improve intrahepatic fat accumulation, we also measured intrahepatic TG (Fig.\u00a0S6B) and blood TG levels (Fig.\u00a0S6C), neither of which showed a significant change. Total intrahepatic CE (Fig.\u00a0S6D) and total plasma CE levels (Fig.\u00a0S6E) were also tested, and there was no significant improvement in either of these results. With respect to NEFAs in the blood, there was also no improvement after FG-4592 injection (Fig.\u00a0S6F). The above results suggest that although FG-4592 may improve liver injury, it does not improve lipid accumulation in the liver.\n\nCDAA-HFD-fed mice were treated with vehicle or FG-4592 for 8 weeks (n\u2009=\u20096 mice/group). A Liver weights. B Ratios of liver mass to body mass. C Serum ALT. D Serum AST. E Representative H&E (up), and Sirius Red (down) staining of liver sections. The circles marked the inflammation foci. n\u2009=\u20093 mice per group, 3 images per mouse. Scale bar is 100\u2009\u03bcm. F The percentage of fibrosis area. G\u2013J Histology scores of hepatic steatosis (G), lobular inflammation (H), ballooning (I), and NAFLD activity (J). K, L Relative mRNA levels of genes related to hepatic inflammation (K) and fibrosis (L). n\u2009=\u20096 biologically independent samples. Data are the means\u2009\u00b1\u2009s.e.m. A\u2013D, F, K, L Statistical analysis was performed using two-tailed Student\u2019s t-tests; G\u2013J Il1b, Il6 in (K), Timp1 and Col5a2 in (L), statistical analysis was performed using two-tailed Mann\u2013Whitney U-tests.\n\nTo further observe liver injury in mice, we made paraffin sections of liver tissue from both groups and stained them with H&E and Sirius red. In the H&E-stained sections, we observed that the degree of steatosis in the livers of the two groups of mice was the same, and therefore, there was no difference in the steatosis score and hepatocyte ballooning\u00a0(Fig.\u00a07G, I). However, there were significantly fewer foci of inflammation than in the vehicle group, and therefore, the score of the lobular inflammation was lower than that of the vehicle group (Fig.\u00a07H). The final calculation of the NASs also showed that FG-4592 injection reduced the symptoms of NASH in mice (Fig.\u00a07J). In Sirius red-stained sections, fibrosis was significantly reduced in the FG-4592-injected group, and this result could be better visualized by counting the fibrosis area proportion (Fig.\u00a07E, F).\n\nAfter FG-4592 injection, inflammation-related genes were significantly downregulated in the mouse liver (Fig.\u00a07K). Additionally, genes related to fibrosis were significantly reduced (Fig.\u00a07L). However, genes related to fatty acid uptake and de novo synthesis were slightly changed, with only the expression level of Fasn being reduced (Fig.\u00a0S6G).\n\nIn conclusion, FG-4592 injection can reduce liver fibrosis and improve NASH symptoms by reducing intrahepatic inflammation.", "section_image": [ "https:////media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_Fig1_HTML.png", "https:////media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_Fig2_HTML.png", "https:////media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_Fig3_HTML.png", "https:////media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_Fig4_HTML.png", "https:////media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_Fig5_HTML.png", "https:////media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_Fig6_HTML.png", "https:////media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41467-024-48954-2/MediaObjects/41467_2024_48954_Fig7_HTML.png" ] }, { "section_name": "Discussion", "section_text": "Chronic liver injury caused by NASH can significantly increase the risk of end-stage liver diseases. However, there is currently no effective drug to treat NASH in the clinical practice. Here, we found that the abundance of So(d18:1) in patients with NASH was significantly increased through metabolomics analysis. So(d18:1) significantly aggravated hepatic lobular inflammation and fibrosis in the livers of NASH model mice. Mechanistically, So(d18:1) inhibits macrophage HIF-2\u03b1 binding with ARNT, thus increasing the secretion of inflammatory factors. This mechanism reveals that macrophage HIF-2\u03b1 may be a potential target for the treatment of NASH. Based on this finding, we tried to use the HIF-2\u03b1 stabilizer FG-4592 to improve NASH, and the results showed that FG-4592 alleviated inflammation and fibrosis in NASH.\n\nLiver steatosis is an early event of NASH. A large amount of lipid accumulation in hepatocytes leads to excessive oxidative stress in hepatocytes, which further induces hepatocyte death, thereby activating inflammation and fibrosis in hepatic lobules4. In NASH patients, we have seen several significant changes in sphingolipids, such as Cer (d18:1/16:0), Cer (d18:1/14:0), Cer (d18:1/20:0), Cer (d18:1/22:0), Cer (d18:1/18:0) and Cer (d18:1/18:0). Their abundances also increased in the serum of NASH patients. Our previous work found that the excessive accumulation of nicotine in the intestine can promote the secretion of intestinal ceramide, thus promoting the progression of NAFLD to NASH6. Other studies also found that increasing liver Cer (d18:1/18:0), and S1P could limit the occurrence and development of chronic inflammation in NAFLD15,16. These studies suggest that sphingolipid metabolism may play an important role in the pathogenesis of NAFLD. However, none of these sphingolipids\u2019 changes could perfectly fit the trend of NASH disease exacerbation and indicate the severity of NASH. But So(d18:1) showed a close relationship to the disease progression of NASH by completely consistent with the trends of the changes in ALT and AST levels representing liver injury. Thus, we think So(d18:1) may be a better indicator of the progression of NASH.\n\nAlthough sphingosine hasn\u2019t been deeply discussed in many studies, there still are some studies that have found the sphingosine\u2019s shape in metabolomics7,8, and they have even found that So(d18:1) in stool can be used as a biomarker to predict cirrhosis9. However, So(d18:1) is usually regarded just as an intermediate product of metabolism between ceramide and S1P, and in-depth mechanistic and functional research is lacking. In this study, we found that So(d18:1) can exist stably in cells at a certain concentration and will not be rapidly converted into ceramide or S1P. Our results showed that So(d18:1) can not only promote the secretion of inflammatory factors in BMDMs but can also aggravate liver inflammation and fibrosis and promote the progression of NASH in animals. Besides, we found that So(d18:1) didn\u2019t directly promote hepatocyte death, nor did it directly induce fibrosis in LX-2 cells. So(d18:1) may still have the possibility of directly acting on other liver cell types. However, myeloid-specific HIF-2\u03b1 overexpression confronted the effects of So(d18:1) on NASH largely (Fig.\u00a05), which could demonstrate the central role of macrophages in this process.\n\nRegarding the origin of the increased circulating So(d18:1) in NASH patients, we examined the amount of So(d18:1) in the whole liver tissue of NASH-modelled mice (Fig.\u00a0S1C). It showed a downward trend within the first two weeks, but no further change in the coming 6 weeks. The metabolism of ceramide is also known to occur in the gut and adipose tissue. There are also results showing that increased levels of So(d18:1) in the feces of NASH-cirrhotic patients, which may serve as one of the biomarkers for predicting NASH-cirrhosis9. This also suggests that the role of microbiota in sphingolipid metabolism should not be underestimated. In summary, the increased So(d18:1) may come from damaged hepatocytes and other organs, especially the gut. Next, we will subsequently measure the levels of So(d18:1) in the gut and adipose tissue of NASH-modelled mice at different time points to further investigate the source of the increased circulating So(d18:1).\n\nHIF is a heterodimer made up of an oxygen-sensitive \u03b1 subunit and a constitutively expressed \u03b2 subunit (ARNT). Under normoxic conditions, HIF-\u03b1 is rapidly hydroxylated and degraded by prolyl hydroxylase (PHD). In contrast, under hypoxia, the activity of prolyl hydroxylase was inhibited, and the HIF protein was stable. HIF-2\u03b1 accumulates and translocates to the nucleus and combines with ARNT to form an active transcription factor complex17. In NASH, HIF-1\u03b1 in macrophages induced by palmitic acid damages autophagic flux and increases IL-1\u03b2 production, aggravating liver injury induced by an MCD diet18. However, the role of HIF-2\u03b1 in macrophages in the progression of NASH is still unclear. In this article, we have validated the role of HIF-2\u03b1 in NASH progression using mice with macrophage-specific knockdown or overexpression of HIF-2\u03b1. Identified that HIF-2\u03b1 is a potential target for intervention in NASH.\n\nRosalistat (FG-4592) is a mature small-molecule drug that is mainly used to treat chronic kidney disease and anaemia, but its role in metabolic diseases has not yet entered clinical trials. In our previous studies, FG-4592 injection was used to improve insulin resistance12. In this study, FG-4592 injection significantly reduced the levels of ALT and AST in the livers of mice, suggesting that the degree of liver injury was reduced. In addition, the expression of genes related to inflammation and fibrosis also decreased. The above results showed that FG-4592 injection can reduce the incidence of NASH. However, many articles have also clarified that the overexpression of HIF-2\u03b1 in liver cells plays a worsening role in insulin resistance and fatty liver19,20. Continuous activation of hepatocyte HIF-2\u03b1 can damage the transcription of fatty acid \u03b2-oxidation-related genes, leading to fat accumulation in the liver20,21. Hepatocyte HIF-2\u03b1 stimulated the production of histidine-rich glycoprotein (HRGP) to activate macrophages to polarize to the M1 type, thus causing liver damage. In our study, the administration of FG-4592 can block the response-ability of proinflammatory macrophages, thus playing a protective role. After FG-4592 reaches the liver, it may indeed lead to the accumulation of lipids in the liver, but it also ensures that hepatocytes damaged by lipotoxicity will not cause further macrophage inflammation. Therefore, from the overall animal experimental data, the administration of FG-4592 still protects the liver from damage in NASH disease. In addition, FG-4592 can also act on other targets, such as adipose tissue HIF-2\u03b1, and promote the production of erythropoietin22,23, which will delay or even improve the disease in many chronic metabolic diseases. Of course, we are also actively seeking ways to improve FG-4592 drug delivery methods, such as using liposome encapsulation to minimize the side effects induced by FG-4592 activation of hepatocyte HIF-2\u03b1.\n\nIn summary, our study found that the active sphingolipid So(d18:1) has a good indicating ability in patients with NASH and that it can bind to HIF-2\u03b1 to aggravate liver inflammation and fibrosis in NASH model mice. Macrophage-specific knockout or overexpression of HIF-2\u03b1 showed that macrophage HIF-2\u03b1 can reduce liver injury and can reduce intrahepatic inflammation and fibrosis. These results not only provide us with a possibility that So(d18:1), a long-chain lipid, binding transcription factor to regulate cellular immune metabolism, but also suggest that the proinflammatory function of So(d18:1) in NASH cannot be ignored. Finally, we used FG-4592 to improve inflammation and fibrosis in NASH. This study provides potential therapeutic targets and strategies for NASH.", "section_image": [] }, { "section_name": "Methods", "section_text": "The use of patient samples in this research complies with the Ethics Committee of Peking University People\u2019s Hospital (Ethics Review Approval No.: 2021PHB124-001). All animal experiments complied with the rules for the use of experimental animals, treatment and euthanasia approved by Peking University Health Science Center (permit: LA2020481).\n\nThe clinical patient cohorts of this study were collected from Peking University People\u2019s Hospital. With the approval of the Ethics Committee of Peking University People\u2019s Hospital (Ethics Review Approval No.: 2021PHB124-001), all volunteers who participated in the study signed a written informed consent form.\n\nThe inclusion criteria were as follows: NASH disease diagnosis was in accordance with the Guidelines of Prevention and Treatment of Non-Alcoholic Fatty Liver Disease: a 2018 Update prepared by the National Workshop on Fatty Liver and Alcoholic Liver Disease, Chinese Society of Hepatology, Chinese Medical Association; Fatty Liver Experts Committee, Chinese Medical Doctor Association. The diagnosis requires the patient to have histological evidence of diffuse hepatocyte steatosis, intrahepatic inflammation and fibrosis, and persistent serum ALT and GGT increases. Patients with alcoholic liver disease, type 3 hepatitis C virus infection, autoimmune hepatitis, hepatolenticular degeneration and drug-induced liver disease were excluded. A FibroScan liver elasticity test was performed to support the diagnosis. All patients were newly diagnosed with NASH and did not receive relevant treatment. Healthy volunteers were also recruited from Peking University People\u2019s Hospital. They were required to have normal serum ALT and GGT levels. FibroScan indicated that their liver elasticity was normal. Their age, sex and BMI were matched to those of NASH patients. This study includes males and females, and the gender of participants was determined based on self-report.\n\nC57BL/6J wild-type male mice were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. Hif2\u03b1fl/fl, Hif2\u03b1LSL/LSL and Lyz2-cre mice which carrying Cre recombinase under the control of the Lyz2 promoter were purchased from Jackson Lab. Hif2\u03b1fl/fl or Hif2\u03b1LSL/LSL mice were bred with Lyz2-cre mice to generate Hif2\u03b1\u0394Lysm mice and LysMHif2\u03b1LSL/LSL mice.\n\nMice were randomly divided into different groups and raised in cages under standard SPF laboratory conditions with free access to water and feed. The temperature was maintained at 21-24 \u00b0C, and the humidity was maintained at 40\u201370%. The light was on from 08:00 to 20:00. The animal use licence number was SYXK (Beijing) 2011-0039. All animal experiments complied with the rules for the use of experimental animals, treatment and euthanasia approved by Peking University Health Science Center (permit: LA2020481).\n\nA normal chow-diet (NCD, 1016706476803973120) was purchased from Beijing Keaoxieli Feed Co., Ltd., in which fat supplies 20% of calories for energy. The GAN diet (D09100310) was purchased from Research Diets, USA, in which fat provides 40% of calories for energy (including palm oil), fructose provides 20% of calories for energy, and 2% cholesterol is added. Mice were fed the GAN diet for 24 weeks to create the NASH model. The CDAA-HFD (A06071302) was purchased from Research Diets, USA, in which fat supplies 60% of calories for energy, and the diet contains 0.1% methionine and does not contain any added choline. Mice were fed the CDAA-HFD for 8 weeks to create the NASH model.\n\nFor the So(d18:1) intraperitoneal injection experiment, the So(d16:1) and So(d18:1) were suspended in 0.5% Carboxymethylcellulose sodium (CMC-Na) solution. 6-week-old male mice were randomly fed the CDAA-HFD for 8 weeks with vehicle (5% CMC-Na), So(d16:1) (10\u2009mg\u2009kg\u22121 body weight) or So(d18:1) (10\u2009mg/kg body weight) injected intraperitoneally every day. For the FG-4592 (MCE, HY-13426) intraperitoneal injection experiment, 6-week-old male mice were randomly fed the CDAA-HFD for 8 weeks with vehicle or FG-4592 (25\u2009mg/kg body weight) injected intraperitoneally every day.\n\nThe HEK293T cell line (catalogue number: GNHu44) and LX-2 cell line (catalogue number: SCSP-527) used in this study were purchased from the National Collection of Authenticated Cell Cultures.\n\nBone marrow-derived macrophages were isolated from the bone marrow of C57BL/6J wild-type mice, macrophage-specific knockout HIF-2\u03b1 mice (Hif2\u03b1\u0394Lysm) and macrophage-specific overexpressing HIF-2\u03b1 mice (LysMHif2\u03b1LSL/LSL).\n\nBMDMs were prepared as previously described24. The bone marrow collected from the femur and tibia of mice was inoculated on sterile petri dishes and cultured in RPMI 1640 containing 10% FBS, 100 units/ml penicillin, 100\u2009mg/ml streptomycin and 10\u2009ng/ml macrophage colony-stimulating factor (M-CSF) for 5\u20136 days. When activating the NLRP3 inflammasome, BMDMs were incubated with LPS (500\u2009ng/ml, 4\u2009h) and then were treated with nigericin (6.7\u2009\u03bcM, 1\u2009h).\n\nAs mentioned earlier25, primary hepatic macrophages were isolated from male mice by injecting type IV collagenase into the liver. Mice were anaesthetized with isoflurane and perfused through the portal vein. Krebs buffer was used to remove blood from the liver. Then, Krebs buffer supplemented with type IV collagenase was used for digestion. After digestion, the liver was collected and rinsed with RPMI 1640. The digested liver cell suspension was passed through a 70-\u03bcm cell filter (BD). The samples were centrifuged at 50\u2009\u00d7\u2009g for 3\u2009min, and the supernatant was retained. The cells were centrifuged at 1200\u2009rpm for 10\u2009min again to precipitate the nonparenchymal cells from the supernatant.\n\nIsolated liver nonparenchymal cells were washed in PBS buffer containing 10% FBS, and red cells were removed. The cells were stained with specific antibody: APC/cy7 anti-CD45 (1:400, BioLegend, 157204, Clone:30-F11), FITC anti-CD11b (1:400, BioLegend, 101205, Clone: M1/70), APC anti-F4/80 (1:400, eBioscience, 17-4801-82, Clone:BM8) at 4\u2009\u00b0C for 30\u2009min protected from light. For intracellular staining, cells were then fixed and permeabilized using the Foxp3 Fixation/Permeabilization kit (eBioscience). Intracellular antibodies were anti-HIF-2\u03b1 PE (1:400, Novus, NB100-122PE, Polyclonal). Cells were then washed with cold PBS 3 times, and analysed by flow cytometry using FACS SORP flow cytometry (BD). The data were analysed using FlowJo software (TreeStar).\n\nCells were seeded into a 48-well plate at a density of 2\u2009\u00d7\u2009104 per well. The luciferase constructs for the HIF response element (HRE), the oxygen-stable HIF-2\u03b1 triple mutant (HIF-2\u03b1\u2122) plasmid and the HIF-1\u03b1TM plasmid (constitutively active HIF-1\u03b1 triple mutants) were previously described12,26. To explore the effect of So(d18:1) on the transcriptional regulatory activity of HIF-2\u03b1, HIF-2\u03b1\u2122 plasmid, p2.1 HRE-Luc plasmid and Renilla positive control plasmid mixed with Lipo8000 transfection reagent were added to each well cells. To explore the effect of So(d18:1) on the transcriptional regulatory activity of HIF-1\u03b1, HIF-1\u03b1\u2122 plasmid, p2.1 HRE-Luc plasmid and Renilla positive control plasmid mixed with Lipo8000 transfection reagent were added to each well cells.\n\nFor the Mammalian Two-Hybrid System, the pG5 luciferase vector was co-transfected with pBIND-HIF-2\u03b1 and pACT-ARNT into cells using the protocol described in the CheckMateTM Mammalian Two-Hybrid System (Promega)14.\n\nFor the mutant assay, HIF-2\u03b1\u2122 plasmid and mHIF-2\u03b1 S304M\u2009+\u2009G323E plasmid were used. For the Cpt1a rHRE binding assay, the pGL3 basic vector (Promega) was cloned with the presumed rHRE1 region in the Cpt1a promoter upstream of the firefly luciferase gene as the reporter plasmid. The reporter plasmid, HIF-2\u03b1\u2122 plasmid or corresponding control empty vector were transfected into HEK293T cells together. The luciferase assay was performed as previously described.\n\nThe cells were treated with a control vehicle, 2\u2009\u03bcM HIF-2\u03b1-specific inhibitor PT2385, and 20\u2009\u03bcM So(d18:1), So(d16:1), Sa(d18:1), So(d20:1) for 24\u2009h, the supernatant was discarded, and the samples were gently rinsed with PBS buffer. Next, 100\u2009\u03bcL of PLB lysis solution was added to the cells, and they were incubated at room temperature for 10\u2009min. Ten microlitres of the cell lysate were added to a white flat-bottomed 96-well plate, and the following procedure was used in the multifunction microplate reader (Tecan): 40\u2009\u03bcL of luciferase substrate was added, the fluorescence value was detected, and 40\u2009\u03bcL of stop liquid was added. Finally, the ratio of the two fluorescence values was calculated.\n\nTargeted lipidomics were performed according to a previous study with minor modifications27. Metabolic analysis of serum samples collected from NASH patients (n\u2009=\u200916) and healthy control (n\u2009=\u200916).Plasma (25\u2009\u03bcL) or Liver tissue (20\u2009mg) was added to 80\u2009\u03bcL of water and homogenized for 1\u2009min. Then, 400\u2009\u03bcL of chloroform and methanol (v/v, 2:1) were added, and the samples were vortexed for 10\u2009min and centrifuged at 4\u2009\u00b0C and 12,000\u2009rpm for 10\u2009min. The lower layer was transferred into a new 1.5-ml tube and dried by a SpeedVac. Subsequently, 100\u2009\u03bcL of cold methanol and isopropanol (v/v, 4:1) was added, and the tubes were vortexed for 10\u2009min and centrifuged at 4\u2009\u00b0C and 18,000\u2009rpm for 10\u2009min. The supernatant was transferred to a vial for MS detection. For plasma (100\u2009\u03bcL), 400\u2009\u03bcL of chloroform and methanol (v/v, 2:1) were added, and the remaining processes were the same as for liver tissue. A Waters UPLC BEH C18 column (2.1\u2009mm (inner diameter)\u2009\u00d7\u2009100\u2009mm (length), 1.7\u2009\u03bcm (particle dimension)) was used for separation. The mobile phase consisted of water (containing 5\u2009mM\u2009ammonium acetate and 0.1% formic acid; phase A) and isopropanol:acetonitrile (1:1, v/v, containing 5\u2009mM ammonium acetate and 0.1% formic acid; phase B) at a flow rate of 0.4\u2009ml/min and a column temperature of 40\u2009\u00b0C, with an injection volume of 2\u2009\u00b5L. The UPLC and MS parameters used were chosen according to a previous study27.\n\nFor the quantification of ceramides, S1P and sphingosine, 25\u2009\u03bcL of plasma or 20\u2009mg of liver tissue was homogenized with 400\u2009\u03bcL of chloroform and methanol (v/v, 2:1) containing 5\u2009\u03bcM sphingosine-d7 d18:1 and 25\u2009\u03bcM ceramide-d7 d18:1/15:0 (Avanti Polar Lipids) as the internal standards. The mixture was oscillated immediately and then centrifuged at 13,000\u2009rpm for 20\u2009min. The lower phase was dried using a SpeedVac. The sediment was dissolved in 100\u2009\u03bcL of isopropanol and acetonitrile (v/v, 1:1) and analysed using the Waters Acquity UPLC coupled with the AB SCIEX QTRAP 5500 system using a Waters UPLC CSH C18 column (3.5\u2009\u03bcm, 2.1\u2009\u00d7\u2009100\u2009mm). The UPLC and MS parameters used were chosen according to a previous study28. The lipid metabolites were quantified using MultiQuant 2.1 (AB SCIEX).\n\nThe NAS, also known as the NAFLD activity score (NAS), is calculated as the sum of three histological components, that is, steatosis (0\u20133), ballooning (0\u20132) and lobular inflammation (0\u20133). Patients with NAS\u2009\u2265\u20095 were considered definite NASH, patients with scores of 3 or 4 were considered borderline NASH, and patients with scores of less than 3 were diagnosed as NAFL.\n\nThe levels of IL-1\u03b2 (Abclonal, RK00006) and IL-18 (Abclonal, RK00104) were measured by ELISA kits according to the manufacturer\u2019s instructions. In short, the standard or sample was added to the antibody-coated plate and incubated at 37\u2009\u00b0C for 120\u2009min. Bio-coupled antibody solution, avidin HRP solution and TMB substrate solution were added to the microporous plate in turn. The absorbance at 450\u2009nm was measured within 15\u2009min after adding the termination solution.\n\nAs for LDH release detection, we use 5\u2009mM APAP and 20\u2009\u03bcM So(d18:1) to treat the primary hepatocytes for 24\u2009h. Collect the supernatant and use the LDH Cytotoxicity Assay Kit (Cat.C0016, Beyotime) to measure the cytotoxicity levels.\n\nWhole-cell lysates were prepared with RIPA buffer. The cell homogenate was incubated on ice in RIPA buffer for 15\u201320\u2009min and then centrifuged at 10,000\u2009rpm at 4\u2009\u00b0C for 10\u2009min. The supernatant was transferred into a new tube and mixed with 5\u00d7 loading buffer. The mixture was boiled for 10\u2009min.\n\nFor co-IP, Protein A/G PLUS agarose beads (Santa Cruz) were placed in the cell lysate supernatant. The samples were incubated upside down overnight at 4\u2009\u00b0C. TBST buffer was used to wash 3 times. Then, 50\u2009\u03bcL of 2\u00d7 loading buffer was added to the beads and boiled for 10\u2009min.\n\nEach well containing 50\u2009\u03bcg of protein lysate was separated by SDS\u2012PAGE, transferred to a nitrocellulose membrane, and immunoblotted at 4\u2009\u00b0C overnight. The antibodies were anti-caspase-1 (1:1000, CST, #24232, Monoclonal), anti-cleaved-caspase-1 (1:1000, CST, #89332, Monoclonal), anti-HIF-2\u03b1 (1:1000, Novus, NB100-132, Clone: ep190b), anti-HIF-1\u03b1 (1:1000, Proteintech, 20960-1-AP, Polyclonal), anti-ARNT (1:1000, Santa Cruz, sc-55526, Monoclonal), anti-GAPDH (1:1000, CST, #5174, Monoclonal) and anti-\u03b2-Actin (1:1000, Abclonal, AC038, Clone: ARC5115-01). The HRP-coupled secondary antibodies used were HRP-conjugated Goat anti-Mouse IgG (H+L) (1:2000, ABclonal, AS003), and HRP-conjugated Goat anti-Rabbit IgG (H+L) (1:2000, ABclonal, AS014) antibodies and immunoblotting was carried out using a chemical imaging system (ChemiDoc, Bio-Rad).\n\nLiver tissues were flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C. Total RNA from frozen liver tissues was extracted using TRIzol reagent (Invitrogen). cDNA was synthesized from 2\u2009\u03bcg of total RNA using 5\u00d7 All-In-One RT MasterMix (Abm). A list of quantitative PCR (qPCR) primer sequences is provided in Supplementary Table\u00a02. The relative amount of each mRNA was compared to the corresponding gene and normalized, and the results are expressed as fold changes relative to the control group.\n\nLibrary preparation and transcriptome sequencing were conducted by GENEWIZ LLC. The Illumina HiSeq platform was used for sequencing. For the data analysis, we first evaluated the quality of the sequence data by fastqc v0.11.9, and the sequence quality was considered to be good for subsequent analysis. Trim-galore v0.6.7 was used for adaptor trimming and low-quality reads. Clean read mapping was conducted by Hisat2 v2.2.1, and we used mm10 as the mouse reference genome. After that, gene expression was quantified by featureCounts v2.0.1. All downstream analyses were performed in R v4.2.1. We used the edgeR v3.38.4 R package for differential expression analysis. We set the cut-off of differentially expressed genes as follows: p-value of 0.05 and absolute value of fold change of 1.5. Gene Ontology (GO) enrichment analysis and transcription factor enrichment analysis were conducted by the clusterProfiler v4.4.4 R package. We used the ARCHS4 transcription factor coexpression database from the Enrichr library as the database for transcription factor enrichment analysis. The record GSE262135 has been submitted to the GEO database.\n\nWe downloaded the count matrix of the GSE166504 dataset from the GEO database and analysed it using R. This is a single-cell transcriptome dataset of livers where mice were fed a chow-diet, an HFHFD diet for 15 weeks, and an HFHFD diet for 30 weeks. We clustered these cells by mRNA expression level using the Seurat package, and then we annotated these cell clusters using the SingleR package.\n\nThis study used GraphPad Prism software v.9.0. and SPSS software v.27.0 for analysis and statistics. The experimental results of this study are presented as the mean\u2009\u00b1\u2009standard error of the mean (SEM). First, the Kolmogorov\u2013Smirnov statistical method was used to detect the normality of all data. If the data conformed to a normal distribution, Student\u2019s t-test was used to compare two groups, one-way ANOVA was used for three or more groups, and Tukey\u2019s post-test was used for statistical analysis. If the data did not conform to a normal distribution, a nonparametric test was used. Mann\u2013Whitney\u2019s statistical method was used for analysis between two groups, and the Kruskal\u2013Wallis and Dunn\u2019s time tests were used for statistical analysis of three or more groups.\n\nFurther information on research design is available in the\u00a0Nature Portfolio Reporting Summary linked to this article.", "section_image": [] }, { "section_name": "Data availability", "section_text": "All of the data supporting the findings of this study are included in the Article and Supplementary information. The RNA-sequencing data generated in this study have been deposited in the GEO database under accession code GSE262135. The data generated in this study are provided in the Supplementary Information/Source Data file. The Single-cell RNA-sequencing data used in this study are available in the GEO database under accession code GSE166504.\u00a0Source data are provided with this paper.", "section_image": [] }, { "section_name": "References", "section_text": "Riazi, K. et al. 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J.X, H.C., X.W., W.C., F.X., Q.N., C.Y., B.Z., M.Z., CY.Y., G.Z., Y.M., Y.W., X.Z., S.Y. and F.L. performed the experiments and analysed the data. Y.P. and H.R. supervised the study. H.C. and J.X. wrote the manuscript with input from all authors. J.L., XM.W., C.Z., P.X., C.J. and L.S. revised the manuscript. J.X, H.C. and X.W. contributed equally to this work. All authors edited the manuscript and approved the final manuscript.\n\nCorrespondence to\n Huiying Rao or Yanli Pang.", "section_image": [] }, { "section_name": "Ethics declarations", "section_text": "Y.M., Y.W. and X.Z. are employees of the Mengniu Institute of Nutrition Science. This study is supported in part by a research fund from the Mengniu Institute of Nutrition Science. The other authors declare no competing interests.", "section_image": [] }, { "section_name": "Peer review", "section_text": "Nature Communications thanks Graeme Lancaster and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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Sphingosine d18:1 promotes nonalcoholic steatohepatitis by inhibiting macrophage HIF-2\u03b1.\n Nat Commun 15, 4755 (2024). https://doi.org/10.1038/s41467-024-48954-2\n\nDownload citation\n\nReceived: 21 June 2023\n\nAccepted: 17 May 2024\n\nPublished: 04 June 2024\n\nVersion of record: 04 June 2024\n\nDOI: https://doi.org/10.1038/s41467-024-48954-2\n\nAnyone you share the following link with will be able to read this content:\n\nSorry, a shareable link is not currently available for this article.\n\n\n\n\n Provided by the Springer Nature SharedIt content-sharing initiative\n ", "section_image": [ "https://data:image/svg+xml;base64,<svg height="81" width="57" xmlns="http://www.w3.org/2000/svg"><g fill="none" fill-rule="evenodd"><path d="m17.35 35.45 21.3-14.2v-17.03h-21.3" fill="#989898"/><path d="m38.65 35.45-21.3-14.2v-17.03h21.3" fill="#747474"/><path d="m28 .5c-12.98 0-23.5 10.52-23.5 23.5s10.52 23.5 23.5 23.5 23.5-10.52 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