Inhibition of Δ24-dehydrocholesterol reductase activates pro-resolving lipid mediator biosynthesis and inflammation resolution Andreas Körnera, Enchen Zhoub, Christoph Müllerc, Yassene Mohammedd, Sandra Hercegc, Franz Bracherc, Patrick C. N. Rensenb, Yanan Wangb, Valbona Mirakaja, and Martin Gierad,1 aDepartment of Anesthesiology and Intensive Care Medicine, Molecular Intensive Care Medicine, Eberhard Karls University Tübingen, 72072 Tübingen, Germany; bDepartment of Medicine, Division of Endocrinology, and Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2333ZA Leiden, The Netherlands; cDepartment of Pharmacy-Center for Drug Research, Ludwig Maximilians University Munich, 81377 Munich, Germany; and dCenter for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands Edited by Christopher K. Glass, University of California San Diego, La Jolla, CA, and approved September 3, 2019 (received for review July 27, 2019) Targeting metabolism through bioactive key metabolites is an desmosterol to cholesterol (Fig. 1A). Of the 10 enzymes involved in upcoming future therapeutic strategy. We questioned how modi- distal cholesterol biosynthesis, starting with squalene, DHCR24 has fying intracellular lipid metabolism could be a possible means for recently taken center stage in several diseases. This enzyme has been alleviating inflammation. Using a recently developed chemical probe linked to Alzheimer’s disease (AD), oncogenic and oxidative stress (SH42), we inhibited distal cholesterol biosynthesis through selective (10), hepatitis C virus (HCV) infections (11), differentiation of T 24 inhibition of Δ -dehydrocholesterol reductase (DHCR24). Inhibition helper-17 cells (12), development of foam cells (13), and prostate of DHCR24 led to an antiinflammatory/proresolving phenotype in a cancer (14). While the role of DHCR24 in AD is controversially murine peritonitis model. Subsequently, we investigated several omics discussed, it has been postulated as a possible drug target for HCV layers in order to link our phenotypic observations with key metabolic infections (11) as well as arteriosclerosis (8). Interestingly, this sug- alterations. Lipidomic analysis revealed a significant increase in endog- gests a pronounced involvement of DHCR24 in inflammatory pro- enous polyunsaturated fatty acid (PUFA) biosynthesis. These data in- cesses, as can be further underlined by the actions of its substrate, tegrated with gene expression analysis, revealing increased expression INFLAMMATION desmosterol. Desmosterol has been shown to interact with the liver IMMUNOLOGY AND of the desaturase Fads6 and the key proresolving enzyme Alox-12/15. X receptors (LXRs), the adenosine 5′-triphosphate–binding cassette Protein array analysis, as well as immune cell phenotype and func- transporter A1 (ABCA1), the sterol response element-binding pro- tional analysis, substantiated these results confirming the antiinflam- γ matory/proresolving phenotype. Ultimately, lipid mediator (LM) analysis tein (SREBP), and ROR (8, 12, 15, 16). In addition, it has been revealed the increased production of bioactive lipids, channeling proven that activation of LXRs has a marked influence on immu- the observed metabolic alterations into a key class of metabolites nological functions and PUFA biosynthesis, particularly within Φ known for their capacity to change the inflammatory phenotype. macrophages (M ) (15, 17). Importantly, PUFAs are substrates for the synthesis of immunologically relevant lipid mediators (LMs) inflammation resolution | desmosterol | cholesterol | lipid mediator | PUFA involved in onset and offset of inflammation (7). n recent years, our knowledge of human lipid metabolism has Significance Isignificantly increased, particularly its role in controlling and defining biological phenotypes. The most important example is Lipid metabolism is crucial to many (patho-)physiological pro- possibly the role of immune cell metabolism in cancer (1). Con- cesses, including inflammation. In particular, cholesterol bio- sequently, this has triggered scientists to develop novel technolo- synthesis has emerged as an exciting novel therapeutic target in gies for studying the interactions between metabolome and numerous recent studies. Much is known about the early steps in phenotype (2). Moreover, metabolic reprogramming has become cholesterol biosynthesis; however, the later steps have hitherto accepted as a possible therapeutic strategy (3, 4). In particular, largely been neglected. Here, we investigated the druggability lipid metabolism has long been recognized for its important role in of distal cholesterol biosynthesis using a selective and potent inflammatory processes (5, 6). However, to date, most therapeutic chemical probe (SH42). This inhibition leads to the accumulation strategies target specific enzymes or receptors rather than attempting of the bioactive metabolite desmosterol, boosting the bio- to comprehensively understand and modify lipid metabolism as a synthesis of polyunsaturated fatty acids (PUFA) and the down- key process for the production and homoeostasis of a plethora of stream production of antiinflammatory mediators. Our report lipid-derived mediators. In this context, we aimed at modifying in- integrates distal cholesterol biosynthesis and its intermediate flammation through global lipid metabolism changes. We hypoth- desmosterol with endogenous PUFA biosynthesis and resolution esize that global changes in lipid metabolism can be controlled in of inflammation, rendering this pathway attractive for further such a way that an intrinsically antiinflammatory/proresolving phe- developments in the context of proresolving therapies. notype is observed. We argue that this would require a shift of lipid Author contributions: P.C.N.R., Y.W., V.M., and M.G. designed research; A.K., E.Z., C.M., metabolism toward the increased production of antiinflammatory and M.G. performed research; S.H., F.B., and M.G. contributed new reagents/analytic and proresolving mediators. As n3-polyunsaturated fatty acids tools; A.K., E.Z., C.M., Y.M., P.C.N.R., Y.W., V.M., and M.G. analyzed data; A.K., E.Z., (PUFAs) have particularly been described as important precursors C.M., Y.M., F.B., P.C.N.R., Y.W., V.M., and M.G. wrote the paper; V.M. contributed to study of several antiinflammatory and proresolving mediators (7), we design; and M.G. guided overall study design. therefore questioned how their endogenous production and me- The authors declare no conflict of interest. tabolism could be boosted, especially during an inflammatory event. This article is a PNAS Direct Submission. Mainly through the recent work of Glass and coworkers (8, 9), Published under the PNAS license. 24 we identified Δ -dehydrocholesterol reductase (DHCR24; also 1To whom correspondence may be addressed. Email: [email protected]. called Seladin-1) as a promising target for controlling lipid metab- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. olism, possibly allowing its reprogramming. DHCR24 is the terminal 1073/pnas.1911992116/-/DCSupplemental. enzyme in cholesterol biosynthesis, catalyzing the transformation of First published September 23, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1911992116 PNAS | October 8, 2019 | vol. 116 | no. 41 | 20623–20634 Downloaded by guest on September 30, 2021 well as the antiinflammatory/proresolving potential, of controlled desmosterol accumulation through selective inhibition of DHCR24 using a recently published, highly in vivo active chemical probe (SH42) (half-maximal inhibitory concentration = 5nM)(21).Ini- tially, we evaluated the cross-reactivity of SH42 with LXRs and a set of additional transcription factors; next, we investigated the probes’ effects on the course of zymosan A (zyA)-induced peritoneal in- flammation. Subsequently, lipidomic analysis was carried out in order to understand the observed phenotypic changes on a molec- ular level. We quantitatively assessed more than 900 lipid species in blood, liver, and peritoneal lavage samples. We monitored serum and peritoneal lavage chemokine and gene expression profiles and conducted protein array analysis. Using tandem mass spectrometry (MS) analysis, we investigated PUFA levels and the production of specialized proresolving mediators (SPMs) and tissue-regenerative mediators. Ultimately, we investigated the therapeutic use of SH42 in animal experiments and investigated functional and phenotypic immune cell responses. Results The Chemical Probe SH42 Does Not Interact Directly with LXRs. To exclude direct LXR activation caused by the employed chemical probe SH42 (Fig. 1), we investigated the regulation of well-known LXR target genes under treatment with this compound. Since cholesterol biosynthesis in cell culture is only active when extracel- lular cholesterol is low (22), we treated RAW264.7 MΦ with high concentrations of SH42 in the presence and absence of fetal calf serum (FCS) as an exogenous source of cholesterol. As can be seen in Fig. 1, the LXR target genes (23) Abca1 and Abcg1 (Srebp1c only at 5 μM) are only activated when no extracellular cholesterol is available. In turn, SH42 blocks cholesterol biosynthesis (21) and causes LXR activation via desmosterol accumulation, but not in a direct fashion; otherwise, target gene regulation would also take place when extracellular cholesterol is available. In order to further strengthen this line of argumentation, we repeated the incubation of Fig. 1.