Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macrophage phenotypes are represented in diseased tissues, but we lack deep understanding of ...mechanisms controlling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derived macrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-induced changes in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions required for Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct gene expression programs and corresponding functions.
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•Myeloid cell diversity in NASH is associated with distinct microanatomical niches•Reprogramming of LXR activity leads to impaired Kupffer cell identify and survival•ATF3 collaborates with LXRs to promote a scar-associated macrophage phenotype•Altered enhancer landscapes enable inference of disease mechanisms
Kupffer cells and recruited myeloid cells contribute to the pathology of nonalcoholic steatohepatitis (NASH), but molecular mechanisms specifying their distinct identities and functions are not known. Seidman and colleagues address this problem by defining cell- and disease-specific enhancer landscapes that enable inference of key transcription factors that drive myeloid cell diversity in NASH.
Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin dryness, inflammation, and itch. A major hallmark of AD is an elevation of the immune cytokines IL-4 and IL-13. ...These cytokines lead to skin barrier disruption and lipid abnormalities in AD, yet the underlying mechanisms are unclear. Sebaceous glands are specialized sebum-producing epithelial cells that promote skin barrier function by releasing lipids and antimicrobial proteins to the skin surface. Here, we show that in AD, IL-4 and IL-13 stimulate the expression of 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), a key rate-limiting enzyme in sex steroid hormone synthesis, predominantly expressed by sebaceous glands in human skin. HSD3B1 enhances androgen production in sebocytes, and IL-4 and IL-13 drive lipid abnormalities in human sebocytes and keratinocytes through HSD3B1. Consistent with our findings in cells, HSD3B1 expression is elevated in the skin of AD patients and can be restored by treatment with the IL-4Rα monoclonal antibody, Dupilumab. Androgens are also elevated in a mouse model of AD, though the mechanism in mice remains unclear. Our findings illuminate a connection between type 2 immunity and sex steroid hormone synthesis in the skin and suggest that abnormalities in sex steroid hormone synthesis may underlie the disrupted skin barrier in AD. Furthermore, targeting sex steroid hormone synthesis pathways may be a therapeutic avenue to restoring normal skin barrier function in AD patients.
Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we ...characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.
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•Determinants of Kupffer cell identity are inferred from dynamic enhancer landscapes•DLL4 activates poised enhancers to induce Kupffer cell lineage-determining factors•LXRα induced by DLL4 drives subsequent activation of Kupffer cell enhancers•TGF-β and desmosterol regulate SMADs and LXRs to maintain Kupffer cell identity
Tissue environment has an instructive role in establishing resident macrophage phenotypes. Sakai et al. provide evidence for a two-step model in which liver-derived signals that include DLL4, TGF-β, and desmosterol induce Kupffer cell (liver-resident macrophage) differentiation by regulating the expression and activities of RBPJ, SMADs, and LXRα.
3-β-hydroxysteroid-Δ
, Δ
-isomerase, known as Emopamil-Binding Protein (EBP), is an endoplasmic reticulum membrane protein involved in cholesterol biosynthesis, autophagy, oligodendrocyte formation. ...The mutation on EBP can cause Conradi-Hunermann syndrome, an inborn error. Interestingly, EBP binds an abundance of structurally diverse pharmacologically active compounds, causing drug resistance. Here, we report two crystal structures of human EBP, one in complex with the anti-breast cancer drug tamoxifen and the other in complex with the cholesterol biosynthesis inhibitor U18666A. EBP adopts an unreported fold involving five transmembrane-helices (TMs) that creates a membrane cavity presenting a pharmacological binding site that accommodates multiple different ligands. The compounds exploit their positively-charged amine group to mimic the carbocationic sterol intermediate. Mutagenesis studies on specific residues abolish the isomerase activity and decrease the multidrug binding capacity. This work reveals the catalytic mechanism of EBP-mediated isomerization in cholesterol biosynthesis and how this protein may act as a multi-drug binder.
Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify ...cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.
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•Primary cilia from diverse organisms and tissues contain oxysterol lipids•Cilia-associated oxysterols bind and activate Smoothened through multiple domains•The oxysterol synthase HSD11β2 generates Smoothened-activating oxysterols•HSD11β2 is enriched in medulloblastoma, and blocking HSD11β2 inhibits cancer growth
How Smoothened localizes to primary cilia and is activated is poorly understood. Raleigh et al. find that cilia contain oxysterol lipids that bind to Smoothened and activate the Hedgehog pathway. Moreover, they identify an enzyme involved in cilia-associated oxysterol biosynthesis that is enriched in Hedgehog pathway-associated medulloblastoma and that blocking this enzyme using a compound in black licorice inhibits tumor growth.
Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. ...However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3β-hydroxysterol Δ
-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45
CD11b
cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)-dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.
An unbiased sample preparation free of interferents (i.e., competing analytes, detergents, plastics) is critical to any lipid MS workflow. Here we present a novel three-phase lipid extraction (3PLE) ...technique using a single-step liquid-liquid extraction (LLE) that allows both extraction and fractionation of lipids by polarity. 3PLE is composed of one aqueous and two organic phases. The upper organic phase is enriched in neutral lipids (triacylglycerols and cholesteryl esters), while the middle organic phase contains the major glycerophospholipids. Thin-layer chromatography, radioactive labeling, and MS were used to confirm lipid partitioning. 3PLE efficiency was demonstrated for bovine liver, human pooled plasma, mouse liver, mouse brain, and mouse white adipose tissue. Compared with the gold-standard Bligh/Dyer LLE, 3PLE showed significant advantages. For direct-infusion workflows, there was a decrease in ion suppression with a corresponding increased number of lipid species identified. For LC/MS workflows, increased signal intensities were observed for lower-abundance lipid species such as phosphatidic acid and phosphatidylserine. 3PLE also proved to be a valuable tool for fatty acid profiling by GC/MS, allowing for the separate identification of neutral and polar fatty acids.
25-Hydroxycholesterol is produced in mammalian tissues. The function of this oxysterol is unknown. Here we describe a central role for 25-hydroxycholesterol in regulating the immune system. In ...initial experiments, we found that stimulation of macrophage Toll-like receptors (TLR) induced expression of cholesterol 25-hydroxylase and the synthesis of 25-hydroxycholesterol. Treatment of naïve B cells with nanomolar concentrations of 25-hydroxycholesterol suppressed IL-2-mediated stimulation of B cell proliferation, repressed activation-induced cytidine deaminase (AID) expression, and blocked class switch recombination, leading to markedly decreased IgA production. Consistent with these findings, deletion of the mouse cholesterol 25-hydroxylase gene caused an increase in serum IgA. Conversely, inactivation of the CYP7B1 oxysterol 7α-hydroxylase, which degrades 25-hydroxycholesterol, decreased serum IgA. The suppression of IgA class switching in B cells by a macrophage-derived sterol in response to TLR activation provides a mechanism for local and systemic negative regulation of the adaptive immune response by the innate immune system.
Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause ...marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia.
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