Oxysterol binding protein regulates the resolution of TLR-induced cytokine production in macrophages.

Toll-like receptors (TLRs) on macrophages sense microbial components and trigger the production of numerous cytokines and chemokines that mediate the inflammatory response to infection. Although many of the components required for the activation of the TLR pathway have been identified, the mechanisms that appropriately regulate the magnitude and duration of the response and ultimately restore homeostasis are less well understood. Furthermore, a growing body of work indicates that TLR signaling reciprocally interacts with other fundamental cellular processes, including lipid metabolism but only a few specific molecular links between immune signaling and the macrophage lipidome have been studied in detail. Oxysterol-binding protein (Osbp) is the founding member of a family of lipid-binding proteins with diverse functions in lipid sensing, lipid transport, and cell signaling but its role in TLR responses is not well defined. Here, we demonstrate that altering the state of Osbp with its natural ligand, 25-hydroxycholesterol (25HC), or pharmacologically, sustains and thereby amplifies Tlr4-induced cytokine production in vitro and in vivo. CRISPR-induced knockdown of Osbp abrogates the ability of these ligands to sustain TLR responses. Lipidomic analysis suggested that the effect of Osbp on TLR signaling may be mediated by alterations in triglyceride production and treating cells with a Dgat1 inhibitor, which blocks triglyceride production and completely abrogates the effect of Osbp on TLR signaling. Thus, Osbp is a sterol sensor that transduces perturbations of the lipidome to modulate the resolution of macrophage inflammatory responses.

Blockade of mTORC1 via Rapamycin Suppresses 27-Hydroxycholestrol-Induced Inflammatory Responses.

Atherosclerosis is characterized by the deposition and accumulation of extracellular cholesterol and inflammatory cells in the arterial blood vessel walls, and 27-hydroxycholesterol (27OHChol) is the most abundant cholesterol metabolite. 27OHChol is an oxysterol that induces immune responses, including immune cell activation and chemokine secretion, although the underlying mechanisms are not fully understood. In this study, we investigated the roles of the mechanistic target of rapamycin (mTOR) in 27HChol-induced inflammation using rapamycin. Treating monocytic cells with rapamycin effectively reduced the expression of CCL2 and CD14, which was involved with the increased immune response by 27OHChol. Rapamycin also suppressed the phosphorylation of S6 and 4EBP1, which are downstream of mTORC1. Additionally, it also alleviates the increase in differentiation markers into macrophage. These results suggest that 27OHChol induces inflammation by activating the mTORC1 signaling pathway, and rapamycin may be useful for the treatment of atherosclerosis-related inflammation involving 27OHchol.

27-Hydroxycholesterol Negatively Affects the Function of Bone Marrow Endothelial Cells in the Bone Marrow.

Hematopoietic stem cells (HSCs) reside in specific microenvironments that facilitate their regulation through both internal mechanisms and external cues. Bone marrow endothelial cells (BMECs), which are found in one of these microenvironments, play a vital role in controlling the self-renewal and differentiation of HSCs during hematological stress. We previously showed that 27-hydroxycholesterol (27HC) administration of exogenous 27HC negatively affected the population of HSCs and progenitor cells by increasing the reactive oxygen species levels in the bone marrow. However, the effect of 27HC on BMECs is unclear. To determine the function of 27HC in BMECs, we employed magnetic-activated cell sorting to isolate CD31+ BMECs and CD31- cells. We demonstrated the effect of 27HC on CD31+ BMECs and HSCs. Treatment with exogenous 27HC led to a decrease in the number of BMECs and reduced the expression of adhesion molecules that are crucial for maintaining HSCs. Our results demonstrate that BMECs are sensitively affected by 27HC and are crucial for HSC survival.

25-hydroxycholesterol promotes brain cytokine production and leukocyte infiltration in a mouse model of lipopolysaccharide-induced neuroinflammation.

Neuroinflammation has been implicated in the pathogenesis of several neurologic and psychiatric disorders. Microglia are key drivers of neuroinflammation and, in response to different inflammatory stimuli, overexpress a proinflammatory signature of genes. Among these, Ch25h is a gene overexpressed in brain tissue from Alzheimer's disease as well as various mouse models of neuroinflammation. Ch25h encodes cholesterol 25-hydroxylase, an enzyme upregulated in activated microglia under conditions of neuroinflammation, that hydroxylates cholesterol to form 25-hydroxycholesterol (25HC). 25HC can be further metabolized to 7α,25-dihydroxycholesterol, which is a potent chemoattractant of leukocytes. We have previously shown that 25HC increases the production and secretion of the proinflammatory cytokine, IL-1ß, by primary mouse microglia treated with lipopolysaccharide (LPS). In the present study, wildtype (WT) and Ch25h-knockout (KO) mice were peripherally administered LPS to induce an inflammatory state in the brain. In LPS-treated WT mice, Ch25h expression and 25HC levels increased in the brain relative to vehicle-treated WT mice. Among LPS-treated WT mice, females produced significantly higher levels of 25HC and showed transcriptomic changes reflecting higher levels of cytokine production and leukocyte migration than WT male mice. However, females were similar to males among LPS-treated KO mice. Ch25h-deficiency coincided with decreased microglial activation in response to systemic LPS. Proinflammatory cytokine production and intra-parenchymal infiltration of leukocytes were significantly lower in KO compared to WT mice. Amounts of IL-1ß and IL-6 in the brain strongly correlated with 25HC levels. Our results suggest a proinflammatory role for 25HC in the brain following peripheral administration of LPS.

27-Hydroxycholesterol Enhances Secretion of Extracellular Vesicles by ROS-Induced Dysregulation of Lysosomes.

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states; they have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, toward secretion as EVs. This altered lysosomal function is likely caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.

18F-Fluorodeoxyglucose positron emission tomography/computed tomography imaging reveals the protective effect of docosahexaenoic acid on glucose metabolism by reducing brain 27-hydroxycholesterol.

Total cholesterol (TC) and the cholesterol oxidation product 27-hydroxycholesterol (27-OHC) are both increased in the elderly. Accumulating evidence has linked 27-OHC to glucose metabolism in the brain, while docosahexaenoic acid (DHA) has been shown to positively regulate the 27-OHC levels. However, it is unclear whether DHA may affect glucose metabolism in the brain by regulating 27-OHC levels. In this study, we hypothesized that DHA supplementation would modulate TC levels and reduce 27-OHC levels, thereby improving brain glucose metabolism in SAMP8 mice. The mice were assigned into the Control group and DHA dietary supplementation group. The study evaluated cholesterol levels, 27-OHC levels, and glucose metabolism in the brain. The results showed that DHA supplementation decreased serum levels of TC, low-density lipoprotein cholesterol (LDL-C), and increased levels of high-density lipoprotein cholesterol (HDL-C); and improved the glucose-corrected standardized uptake value of cortex, hippocampus, and whole brain regions in SAMP8 mice. In conclusion, supplementation of DHA could regulate the cholesterol composition and reduce the level of 27-OHC, thereby improving brain glucose metabolism in SAMP8 mice.

Role of Cholesterol Metabolic Enzyme CYP46A1 and Its Metabolite 24S-Hydroxycholesterol in Ischemic Stroke.

BACKGROUND: For several decades, it has been recognized that overactivation of the glutamate-gated N-methyl-D-aspartate receptors (NMDARs) and subsequent Ca2+ toxicity play a critical role in ischemic brain injury. 24S-hydroxycholesterol (24S-HC) is a major cholesterol metabolite in the brain, which has been identified as a potent positive allosteric modulator of NMDAR in rat hippocampal neurons. We hypothesize that 24S-HC worsens ischemic brain injury via its potentiation of the NMDAR, and reducing the production of 24S-HC by targeting its synthetic enzyme CYP46A1 provides neuroprotection. METHODS: We tested this hypothesis using electrophysiological, pharmacological, and transgenic approaches and in vitro and in vivo cerebral ischemia models. RESULTS: Our data show that 24S-HC potentiates NMDAR activation in primary cultured mouse cortical neurons in a concentration-dependent manner. At 10 µmol/L, it dramatically increases the steady-state currents by 51% and slightly increases the peak currents by 20%. Furthermore, 24S-HC increases NMDA and oxygen-glucose deprivation-induced cortical neuronal injury. The increased neuronal injury is largely abolished by NMDAR channel blocker MK-801, suggesting an NMDAR-dependent mechanism. Pharmacological inhibition of CYP46A1 by voriconazole or gene knockout of Cyp46a1 dramatically reduces ischemic brain injury. CONCLUSIONS: These results identify a new mechanism and signaling cascade that critically impacts stroke outcome: CYP46A1 → 24S-HC → NMDAR → ischemic brain injury. They offer proof of principle for further development of new strategies for stroke intervention by targeting CYP46A1 or its metabolite 24S-HC.

Mutant induced neurons and humanized mice enable identification of Niemann-Pick type C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

25-Hydroxycholesterol induces oxidative stress, leading to apoptosis and ferroptosis in extravillous trophoblasts.

25-hydroxycholesterol (25HC) is an oxysterol derived from cholesterol and plays a role in various cellular processes, such as lipid metabolism, inflammatory responses, and cell survival. Extravillous trophoblasts (EVTs) are a major cell type found in the placenta, which are highly energetic cells with proliferative and invasive properties. EVT dysfunction can lead to pregnancy complications, including preeclampsia and intrauterine growth restriction. This study investigated the effects and underlying mechanisms of action of 25HC on EVT proliferation. Swan 71 cells, an EVT cell line, were treated with different concentrations of 25HC. Next, cell proliferation was assessed. The mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potentials (MMPs), lipid peroxidation (LPO), and glutathione (GSH) levels were measured. Apoptosis, ferroptosis, and autophagy were evaluated by western blotting and flow cytometry. The results revealed that 25HC significantly inhibited proliferation and decreased the metabolic activity of EVTs. Moreover, 25HC caused oxidative stress by altering mtROS, LPO, MMPs, and GSH levels. Additionally, 25HC induces apoptosis, ferroptosis, and autophagy through the modulation of relevant protein levels. Interestingly, pretreatment with Z-VAD-FMK, an apoptosis inhibitor, and ferrostatin-1, a ferroptosis inhibitor, partially restored the effects of 25HC on cell proliferation, oxidative stress, and cell death. In summary, our findings suggest that 25HC treatment inhibits EVT proliferation and triggers apoptosis, ferroptosis, and autophagy, which are attributable to oxidative stress.

Oxysterol Induces Expression of 60 kDa Chaperone Protein on Cell Surface of Microglia.

Microglia, essential immune cells in the brain, play crucial roles in neuroinflammation by performing various functions such as neurogenesis, synaptic pruning, and pathogen defense. These cells are activated by inflammatory factors like ß-amyloid (Aß) and oxysterols, leading to morphological and functional changes, including the secretion of inflammatory cytokines and the upregulation of MHC class II molecules. This study focused on identifying specific markers for microglial activation, with a particular emphasis on the roles of oxysterols in this process. We used the HMC3 human microglial cell line to investigate the induction of heat shock protein 60 (HSP60), a chaperonin protein by oxysterols, specifically in the presence of 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol). Our findings obtained by the proteomics approach revealed that these oxysterols significantly increased HSP60 expression on microglial cells. This induction was further confirmed using Western blot analysis and immunofluorescence microscopy. Additionally, Aß1-42 also promoted HSP60 expression, indicating its role as a microglial activator. HSP60 involved in protein folding and immune modulation was identified as a potential marker for microglial activation. This study underscores the importance of HSP60 in the inflammatory response of microglia, suggesting its utility as a target for new therapeutic approaches in neuroinflammatory diseases such as Alzheimer's disease (AD).

25-Hydroxycholesterol attenuates tumor necrosis factor alpha-induced blood-brain barrier breakdown in vitro.

Intracellular cholesterol metabolism is regulated by the SREBP-2 and LXR signaling pathways. The effects of inflammation on these molecular mechanisms remain poorly studied, especially at the blood-brain barrier (BBB) level. Tumor necrosis factor α (TNFα) is a proinflammatory cytokine associated with BBB dysfunction. Therefore, the aim of our study was to investigate the effects of TNFα on BBB cholesterol metabolism, focusing on its underlying signaling pathways. Using a human in vitro BBB model composed of human brain-like endothelial cells (hBLECs) and brain pericytes (HBPs), we observed that TNFα increases BBB permeability by degrading the tight junction protein CLAUDIN-5 and activating stress signaling pathways in both cell types. TNFα also promotes cholesterol release and decreases cholesterol accumulation and APOE secretion. In hBLECs, the expression of SREBP-2 targets (LDLR and HMGCR) is increased, while ABCA1 expression is decreased. In HBPs, only LDLR and ABCA1 expression is increased. TNFα treatment also induces 25-hydroxycholesterol (25-HC) production, a cholesterol metabolite involved in the immune response and intracellular cholesterol metabolism. 25-HC pretreatment attenuates TNFα-induced BBB leakage and partially alleviates the effects of TNFα on ABCA1, LDLR, and HMGCR expression. Overall, our results suggest that TNFα favors cholesterol efflux via an LXR/ABCA1-independent mechanism at the BBB, while it activates the SREBP-2 pathway. Treatment with 25-HC partially reversed the effect of TNFα on the LXR/SREBP-2 pathways. Our study provides novel perspectives for better understanding cerebrovascular signaling events linked to BBB dysfunction and cholesterol metabolism in neuroinflammatory diseases.

ATP6V0A1-dependent cholesterol absorption in colorectal cancer cells triggers immunosuppressive signaling to inactivate memory CD8+ T cells.

Obesity shapes anti-tumor immunity through lipid metabolism; however, the mechanisms underlying how colorectal cancer (CRC) cells utilize lipids to suppress anti-tumor immunity remain unclear. Here, we show that tumor cell-intrinsic ATP6V0A1 drives exogenous cholesterol-induced immunosuppression in CRC. ATP6V0A1 facilitates cholesterol absorption in CRC cells through RAB guanine nucleotide exchange factor 1 (RABGEF1)-dependent endosome maturation, leading to cholesterol accumulation within the endoplasmic reticulum and elevated production of 24-hydroxycholesterol (24-OHC). ATP6V0A1-induced 24-OHC upregulates TGF-ß1 by activating the liver X receptor (LXR) signaling. Subsequently, the release of TGF-ß1 into the tumor microenvironment by CRC cells activates the SMAD3 pathway in memory CD8+ T cells, ultimately suppressing their anti-tumor activities. Moreover, we identify daclatasvir, a clinically used anti-hepatitis C virus (HCV) drug, as an ATP6V0A1 inhibitor that can effectively enhance the memory CD8+ T cell activity and suppress tumor growth in CRC. These findings shed light on the potential for ATP6V0A1-targeted immunotherapy in CRC.

Efficient production of 22(R)-hydroxycholesterol via combination optimization of Saccharomyces cerevisiae.

22(R)-hydroxycholesterol (22(R)-HCHO) is a crucial precursor of steroids biosynthesis with various biological functions. However, the production of 22(R)-HCHO is expensive and unsustainable due to chemical synthesis and extraction from plants or animals. This study aimed to construct a microbial cell factory to efficiently produce 22(R)-HCHO through systems metabolic engineering. First, we tested 7-dehydrocholesterol reductase (Dhcr7s) and cholesterol C22-hydroxylases from different sources in Saccharomyces cerevisiae, and the titer of 22(R)-HCHO reached 128.30 mg L-1 in the engineered strain expressing Dhcr7 from Columba livia (ClDhcr7) and cholesterol 22-hydroxylase from Veratrum californicum (VcCyp90b27). Subsequently, the 22(R)-HCHO titer was significantly increased to 427.78 mg L-1 by optimizing the critical genes involved in 22(R)-HCHO biosynthesis. Furthermore, hybrid diploids were constructed to balance cell growth and 22(R)-HCHO production and to improve stress tolerance. Finally, the engineered strain produced 2.03 g L-1 of 22(R)-HCHO in a 5-L fermenter, representing the highest 22(R)-HCHO titer reported to date in engineered microbial cell factories. The results of this study provide a foundation for further applications of 22(R)-HCHO in various industrially valuable steroids.

27-Hydroxycholesterol acts on estrogen receptor α expressed by POMC neurons in the arcuate nucleus to modulate feeding behavior.

Oxysterols are metabolites of cholesterol that regulate cholesterol homeostasis. Among these, the most abundant oxysterol is 27-hydroxycholesterol (27HC), which can cross the blood-brain barrier. Because 27HC functions as an endogenous selective estrogen receptor modulator, we hypothesize that 27HC binds to the estrogen receptor α (ERα) in the brain to regulate energy balance. Supporting this view, we found that delivering 27HC to the brain reduced food intake and activated proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (POMCARH) in an ERα-dependent manner. In addition, we observed that inhibiting brain ERα, deleting ERα in POMC neurons, or chemogenetic inhibition of POMCARH neurons blocked the anorexigenic effects of 27HC. Mechanistically, we further revealed that 27HC stimulates POMCARH neurons by inhibiting the small conductance of the calcium-activated potassium (SK) channel. Together, our findings suggest that 27HC, through its interaction with ERα and modulation of the SK channel, inhibits food intake as a negative feedback mechanism against a surge in circulating cholesterol.

Effect of compound treatments on mouse lens viscoelasticity.

Previous studies have shown that pharmaceutical agents such as lipoic acid have the ability to soften the lens, presenting a promising avenue for treating presbyopia. One obstacle encountered in the preclinical stage of such agents is the need for precise measurements of lens elasticity in experimental models. This study aimed to evaluate the effects of 25-hydroxycholesterol, lipoic acid, and obeticholic acid on the viscoelastic properties of mouse lenses using a custom-built elastometer system. Data were acquired on lenses from C57BL/6J female mice from two age groups: young (age: 8-10 weeks) and old (age: 32-43 weeks). OD lenses were used as the control and OS lenses were treated. Control lenses were immersed in Dulbecco's Modified Eagle Medium (DMEM) and treatment lenses were immersed in a compound solution containing 25-hydroxycholesterol (5 young and 5 old), lipoic acid at 2.35 mM (5 young and 5 old), lipoic acid at 0.66 mM (5 old), or obeticholic acid (5 old) at 37 °C for 18 h. After treatment, the mouse lenses were placed in a DMEM-filled chamber within a custom-built elastometer system that recorded the load and lens shape as the lens was compressed by 600 µm at a speed of 50 µm/s. The load was continuously recorded during compression and during stress-relaxation. The compression phase was fit with a linear function to quantify lens stiffness. The stress-relaxation phase was fit with a 3-term exponential relaxation model providing relaxation time constants (t1, t2, t3), and equilibrium load. The lens stiffness, time constants and equilibrium load were compared for the control and treated groups. Results revealed an increase in stiffness with age for the control group (young: 1.16 ± 0.11 g/mm, old: 1.29 ± 0.14 g/mm) and relaxation time constants decreased with age (young: t1 = 221.9 ± 29.0 s, t2 = 24.7 ± 3.8 s, t3 = 3.12 ± 0.87 s, old: t1 = 183.0 ± 22.0 s, t2 = 20.6 ± 2.6 s and t3 = 2.24 ± 0.43 s). Among the compounds tested, only 25-hydroxycholesterol produced statistically significant changes in the lens stiffness, relaxation time constants, and equilibrium load. In conclusion, older mouse lenses are stiffer and less viscous than young mouse lenses. Notably, no significant change in lens stiffness was observed following treatment with lipoic acid, contrary to previous findings.

A rapid quantitative UPLC-MS/MS method for analysis of key regulatory oxysterols in biological samples for liver cancer.

An UPLC-APCI-MS/MS method was developed for the simultaneous determination of cholesterol, 7-dehydrocholesterol (7DHC) and eight oxysterols including 27-hydroxycholesterol (27OHC), 7α-hydroxycholesterol (7αOHC), 7ß-hydroxycholesterol (7ßOHC), 24S-hydroxycholesterol (24SOHC), 25-hydroxycholesterol (25OHC), 7α,24S-dihydroxycholesterol (7α,24SdiOHC), 7α,25-dihydroxycholesterol (7α,25diOHC), and 7α,27-dihydroxycholesterol (7α,27diOHC). It has been used for quantitative analysis of cholesterol, 7DHC and eight oxysterols in hepatocellular carcinoma (HCC) cells, plasma and tumor tissue samples. And the above compounds were extracted from the biological matrix (plasma and tissue) using liquid-liquid extraction with hexane/isopropanol after saponification to cleave the steroids from their esterified forms without further derivatization. Then cholesterol, 7DHC and oxysterols were separated on a reversed phase column (Agilent Zorbax Eclipse plus, C18) within 8 min using a gradient elution with 0.1 % formic acid in H2O and methanol and detected by an APCI triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) of the cholesterol, 7DHC and oxysterols ranged from 3.9 ng/mL to 31.25 ng/mL, and the recoveries ranged from 83.0 % to 113.9 %. Cholesterol, 7DHC and several oxysterols including 27OHC, 7αOHC and 7ßOHC were successfully quantified in HCC cells, plasma, tissues and urine of HCC mice. Results showed that 27OHC was at high levels in three kind of HCC cells and tumor tissues as well as plasma samples from both HepG2 and Huh7 bearing mice model，and the high levels of 27OHC in tumors were associated with HCC development. Moreover, the levels of cholesterol in HCC cells and tumor issues varied in different HCC cells and mice model. Oxysterols profiling in biological samples might provide complementary information in cancer diagnosis.

1ß-Hydroxydeoxycholic Acid as an Endogenous Biomarker in Human Plasma for Assessment of CYP3A Clinical Drug-Drug Interaction Potential.

4ß-Hydroxycholesterol (4ß-HC) in plasma has been used as a biomarker to assess CYP3A drug-drug interaction (DDI) potential during drug development. However, due to the long half-life and narrow dynamic range of 4ß-HC, its use has been limited to the identification of CYP3A inducers, but not CYP3A inhibitors. The formation of 1ß-hydroxydeoxycholic acid (1ß-OH DCA) from deoxycholic acid (DCA) is mediated by CYP3A, thus 1ß-OH DCA can potentially serve as an alternative to 4ß-HC for assessment of CYP3A DDI potential. To study this feasibility, we developed a sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of 1ß-OH DCA and its glycine and taurine conjugates in human plasma with the lower limit of quantitation of 50 pg/ml, which enabled the quantitation of basal levels and further reduction. The method was applied to a DDI study to assess how 1ß-OH DCA and its glycine and taurine conjugates would respond to CYP3A induction or inhibition. Rifampin induction resulted in an increase of 1ß-OH DCA and its conjugates in plasma, with 6.8-, 7.8-, 8.3-, and 10.3-fold increases of area under the curve from the time of dosing to the last measurable concentration (AUCLST), area under the curve from the time of dosing to 24 hours (AUC24h), C max, and mean concentrations for total 1ß-OH DCA (total of all three forms), respectively. Importantly, inhibition with itraconazole resulted in notable reduction of these biomarkers, with 84%, 85%, 82%, and 81% reductions of AUCLST, AUC24h, C max, and mean concentrations for total 1ß-OH DCA, respectively. These preliminary data demonstrate for the first time that total 1ß-OH DCA in plasma has the potential to serve as a biomarker for CYP3A DDI assessment in early clinical development and may provide key advantages over 4ß-HC. SIGNIFICANCE STATEMENT: The authors have reported the use of total 1ß-hydroxydeoxycholic acid (1ß-OH DCA) (sum of 1ß-OH DCA and its glycine and taurine conjugates) plasma exposure as a biomarker for CYP3A activity. Itraconazole inhibition led to an 81%-85% decrease of total 1ß-OH DCA plasma exposures, whereas rifampin induction led to a 6.8- to 10.3-fold increase of total 1ß-OH DCA plasma exposures. Using 1ß-OH DCA exposures in plasma also provides the benefit of allowing pharmacokinetic and biomarker assessment using the same matrix.

Cholesterol profiling reveals 7ß-hydroxycholesterol as a pathologically relevant peripheral biomarker of Alzheimer's disease.

AIM: Cholesterol homeostasis is associated with Alzheimer's disease (AD). Despite the multitude of cholesterol metabolites, little is known about which metabolites are directly involved in AD pathogenesis and can serve as its potential biomarkers. METHODS: To identify "hit" metabolites, steroid profiling was conducted in mice with different age, diet, and genotype and also in humans with normal cognition, mild cognitive impairment, and AD using gas chromatography-mass spectrometry. Then, using one of the "hit" molecules (7ß-hydroxycholesterol; OHC), molecular and histopathological experiment and behavioral testing were conducted in normal mice following its intracranial stereotaxic injection to see whether this molecule drives AD pathogenesis and causes cognitive impairment. RESULTS: The serum levels of several metabolites, including 7ß-OHC, were increased by aging in the 3xTg-AD unlike normal mice. Consistently, the levels of 7ß-OHC were increased in the hairs of patients with AD and were correlated with clinical severity. We found that 7ß-OHC directly affects AD-related pathophysiology; intrahippocampal injection of 7ß-OHC induced astrocyte and microglial cell activation, increased the levels of pro-inflammatory cytokines (TNF-alpha, IL-1ß, IL-6), and enhanced amyloidogenic pathway. Mice treated with 7ß-OHC also exhibited deficits in memory and frontal/executive functions assessed by object recognition and 5-choice serial reaction time task, respectively. CONCLUSIONS: Our results suggest that 7ß-OHC could serve as a convenient, peripheral biomarker of AD. As directly involved in AD pathogenesis, 7ß-OHC assay may help actualize personalized medicine in a way to identify an at-risk subgroup as a candidate population for statin-based AD treatment.

Neurosteroids mediate and modulate the effects of pro-inflammatory stimulation and toll-like receptors on hippocampal plasticity and learning.

Pro-inflammatory changes contribute to multiple neuropsychiatric illnesses. Understanding how these changes are involved in illnesses and identifying strategies to alter inflammatory responses offer paths to potentially novel treatments. We previously found that acute pro-inflammatory stimulation with high (µg/ml) lipopolysaccharide (LPS) for 10-15 min dampens long-term potentiation (LTP) in the hippocampus and impairs learning. Effects of LPS involved non-canonical inflammasome signaling but were independent of toll-like receptor 4 (TLR4), a known LPS receptor. Low (ng/ml) LPS also inhibits LTP when administered for 2-4 h, and here we report that this LPS exposure requires TLR4. We also found that effects of low LPS on LTP involve the oxysterol, 25-hydroxycholesterol, akin to high LPS. Effects of high LPS on LTP are blocked by inhibiting synthesis of 5α-reduced neurosteroids, indicating that neurosteroids mediate LTP inhibition. 5α-Neurosteroids also have anti-inflammatory effects, and we found that exogenous allopregnanolone (AlloP), a key 5α-reduced steroid, prevented effects of low but not high LPS on LTP. We also found that activation of TLR2, TLR3 and TLR7 inhibited LTP and that AlloP prevented the effects of TLR2 and TLR7, but not TLR3. The enantiomer of AlloP, a steroid that has anti-inflammatory actions but low activity at GABAA receptors, prevented LTP inhibition by TLR2, TLR3 and TLR7. In vivo, both AlloP enantiomers prevented LPS-induced learning defects. These studies indicate that neurosteroids play complex roles in network effects of acute neuroinflammation and have potential importance for development of AlloP analogues as therapeutic agents.

Transient hydroxycholesterol treatment restrains TCR signaling to promote long-term immunity.

T cell receptor (TCR) plays a fundamental role in adaptive immunity, and TCR-T cell therapy holds great promise for treating solid tumors and other diseases. However, there is a noticeable absence of chemical tools tuning TCR activity. In our study, we screened natural sterols for their regulatory effects on T cell function and identified 7-alpha-hydroxycholesterol (7a-HC) as a potent inhibitor of TCR signaling. Mechanistically, 7a-HC promoted membrane binding of CD3ε cytoplasmic domain, a crucial signaling component of the TCR-CD3 complex, through alterations in membrane physicochemical properties. Enhanced CD3ε membrane binding impeded the condensation between CD3ε and the key kinase Lck, thereby inhibiting Lck-mediated TCR phosphorylation. Transient treatments of TCR-T cells with 7a-HC resulted in reduced signaling strength, increased memory cell populations, and superior long-term antitumor functions. This study unveils a chemical regulation of TCR signaling, which can be exploited to enhance the long-term efficacy of TCR-T cell therapy.