The Lipidomics Reporting Checklist A framework for transparency of lipidomic experiments and repurposing resource data.

The rapid increase in lipidomic studies has led to a collaborative effort within the community to establish standards and criteria for producing, documenting, and disseminating data. Creating a dynamic easy-to-use checklist that condenses key information about lipidomic experiments into common terminology will enhance the field's consistency, comparability, and repeatability. Here, we describe the structure and rationale of the established Lipidomics Minimal Reporting Checklist to increase transparency in lipidomics research.

Female sex hormones exacerbate retinal neurodegeneration.

Neurodegenerative disorders such as Alzheimer's disease and macular degeneration represent major sources of human suffering, yet the factors influencing disease severity remain poorly understood. Sex has been implicated as one potential modifying factor. Here, we show that female sex is a risk factor for worsened outcomes in a model of retinal degeneration. Further, we show that this susceptibility is caused by the presence of female-specific circulating sex hormones. The adverse effect of female sex hormones was specific to diseased retinal neurons, and depletion of these hormones ameliorated this phenotypic effect. These findings provide novel insights into the pathogenesis of neurogenerative diseases and how sex hormones can impact the severity of disease. These findings have far-reaching implications for clinical trial design and the use of hormonal therapy in females with certain neurogenerative disorders.

Interaction between the gut microbiota and colonic enteroendocrine cells regulates host metabolism.

Nutrient handling is an essential function of the gastrointestinal tract. Hormonal responses of small intestinal enteroendocrine cells (EECs) have been extensively studied but much less is known about the role of colonic EECs in metabolic regulation. To address this core question, we investigated a mouse model deficient in colonic EECs. Here we show that colonic EEC deficiency leads to hyperphagia and obesity. Furthermore, colonic EEC deficiency results in altered microbiota composition and metabolism, which we found through antibiotic treatment, germ-free rederivation and transfer to germ-free recipients, to be both necessary and sufficient for the development of obesity. Moreover, studying stool and blood metabolomes, we show that differential glutamate production by intestinal microbiota corresponds to increased appetite and that colonic glutamate administration can directly increase food intake. These observations shed light on an unanticipated host-microbiota axis in the colon, part of a larger gut-brain axis, that regulates host metabolism and body weight.

Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating Jmjd3.

Statins are known to be anti-inflammatory, but the mechanism remains poorly understood. Here, we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the adenosine triphosphate (ATP) synthase in the inner mitochondrial membrane and changes the proton gradient in the mitochondria. This activates nuclear factor kappa-B (NF-κB) and Jmjd3 expression, which removes the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus lipopolysaccharide (M1), macrophages, either treated with statins in vitro or isolated from statin-fed mice, express lower levels proinflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL-4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension.

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease.

LPD-3 as a megaprotein brake for aging and insulin-mTOR signaling in C. elegans.

Insulin-mechanistic target of rapamycin (mTOR) signaling drives anabolic growth during organismal development; its late-life dysregulation contributes to aging and limits lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here, we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin, INS-7, is drastically overproduced from early life and shortens lifespan in lpd-3 mutants. LPD-3 forms a bridge-like tunnel megaprotein to facilitate non-vesicular cellular lipid trafficking. Lipidomic profiling reveals increased hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1. Reducing the abundance of HYL-1, insulin receptor/DAF-2 or mTOR/LET-363, normalizes INS-7 levels and rescues the lifespan of lpd-3 mutants. LPD-3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age. We propose that LPD-3 acts as a megaprotein brake for organismal aging and that its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

Assessment of the Cutaneous Hormone Landscapes and Microbiomes in Vulvar Lichen Sclerosus.

Vulvar lichen sclerosus (VLS) is a progressive skin disease of unknown etiology. In this longitudinal case-control exploratory study, we evaluated the hormonal and microbial landscapes in 18 postmenopausal females (mean [SD] age: 64.4 [8.4] years) with VLS and controls. We reevaluated the patients with VLS after 10-14 weeks of daily topical class I steroid. We found that groin cutaneous estrone was lower in VLS than in controls (-22.33, 95% confidence interval [CI] = -36.96 to -7.70; P = .006); cutaneous progesterone was higher (5.73, 95% CI = 3.74-7.73; P < .0001). Forehead 11-deoxycortisol (-0.24, 95% CI = -0.42 to -0.06; P = .01) and testosterone (-7.22, 95% CI = -12.83 to -1.62; P = .02) were lower in disease. With treatment, cutaneous estrone (-7.88, 95% CI = -44.07 to 28.31; P = .62), progesterone (2.02, 95% CI = -2.08 to 6.11; P = .29), and 11-deoxycortisol (-0.13, 95% CI = -0.32 to 0.05; P = .15) normalized; testosterone remained suppressed (-7.41, 95% CI = -13.38 to -1.43; P = .02). 16S ribosomal RNA V1-V3 and ITS1 amplicon sequencing revealed bacterial and fungal microbiome alterations in disease. Findings suggest that cutaneous sex hormone and bacterial microbiome alterations may be associated with VLS in postmenopausal females.

Paraoxonase-like APMAP maintains endoplasmic reticulum-associated lipid and lipoprotein homeostasis.

Oxidative stress perturbs lipid homeostasis and contributes to metabolic diseases. Though ignored compared to mitochondrial oxidation, the endoplasmic reticulum (ER) generates reactive oxygen species requiring antioxidant quality control. Using multi-organismal profiling featuring Drosophila, zebrafish, and mammalian cells, here we characterize the paraoxonase-like APMAP as an ER-localized protein that promotes redox and lipid homeostasis and lipoprotein maturation. APMAP-depleted mammalian cells exhibit defective ER morphology, elevated ER and oxidative stress, lipid droplet accumulation, and perturbed ApoB-lipoprotein homeostasis. Critically, APMAP loss is rescued with chemical antioxidant NAC. Organismal APMAP depletion in Drosophila perturbs fat and lipoprotein homeostasis, and zebrafish display increased vascular ApoB-containing lipoproteins, particles that are atherogenic in mammals. Lipidomics reveals altered polyunsaturated phospholipids and increased ceramides upon APMAP loss, which perturbs ApoB-lipoprotein maturation. These ApoB-associated defects are rescued by inhibiting ceramide synthesis. Collectively, we propose APMAP is an ER-localized antioxidant that promotes lipid and lipoprotein homeostasis.

DGAT2 inhibition blocks SREBP-1 cleavage and improves hepatic steatosis by increasing phosphatidylethanolamine in the ER.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage in vivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.

Adipose-specific overexpression of human AGPAT2 in mice causes increased adiposity and mild hepatic dysfunction.

AGPAT2, a critical enzyme involved in the biosynthesis of phospholipids and triacylglycerol (TAG), is highly expressed in adipose tissue (AT). Whether overexpression of AGPAT2 in AT will result in increased TAG synthesis (obesity) and its metabolic complications remains unknown. We overexpressed human AGPAT2 specifically in AT using the adiponectin promoter and report increased mass of subcutaneous, gonadal, and brown AT in wild-type mice. Unexpectedly, overexpression of hAGPAT2 did not change the pattern of phospholipid or TAG concentration of the AT depots. Although there is an increase in liver weight, plasma aspartate aminotransferase, and plasma insulin at various time points of the study, it did not result in significant liver dysfunction. Despite increased adiposity in the Tg-AT-hAGPAT2;mAgpat2+/+ mice, there was no significant increase in TAG concentration of AT. Therefore, this study suggests a role of AGPAT2 in the generation of AT, but not for adipocyte TAG synthesis.

CLSTN3B enhances adipocyte lipid droplet structure and function via endoplasmic reticulum contact.

Interorganelle contacts facilitate material exchanges and sustain the structural and functional integrity of organelles. Lipid droplets (LDs) of adipocytes are responsible for energy storage and mobilization responding to body needs. LD biogenesis defects compromise the lipid-storing capacity of adipocytes, resulting in ectopic lipid deposition and metabolic disorders, yet how the uniquely large LDs in adipocytes attain structural and functional maturation is incompletely understood. Here we show that the mammalian adipocyte-specific protein CLSTN3B is crucial for adipocyte LD maturation. CLSTN3B employs an arginine-rich segment to promote extensive contact and hemifusion-like structure formation between the endoplasmic reticulum (ER) and LD, allowing ER-to-LD phospholipid diffusion during LD expansion. CLSTN3B ablation results in reduced LD surface phospholipid density, increased turnover of LD-surface proteins, and impaired LD functions. Our results establish the central role of CLSTN3B in the adipocyte-specific LD maturation pathway that enhances lipid storage and maintenance of metabolic health under caloric overload.

DHCR24-mediated sterol homeostasis during spermatogenesis is required for sperm mitochondrial sheath formation and impacts male fertility over time.

Desmosterol and cholesterol are essential lipid components of the sperm plasma membrane. Cholesterol efflux is required for capacitation, a process through which sperm acquire fertilizing ability. In this study, using a transgenic mouse model overexpressing 24-dehydrocholesterol reductase (DHCR24), an enzyme in the sterol biosynthesis pathway responsible for the conversion of desmosterol to cholesterol, we show that disruption of sterol homeostasis during spermatogenesis led to defective sperm morphology characterized by incomplete mitochondrial packing in the midpiece, reduced sperm count and motility, and a decline in male fertility with increasing paternal age, without changes in body fat composition. Sperm depleted of desmosterol exhibit inefficiency in the acrosome reaction, metabolic dysfunction, and an inability to fertilize the egg. These findings provide molecular insights into sterol homeostasis for sperm capacitation and its impact on male fertility.

Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating JMJD3.

Stains are known to be anti-inflammatory, but the mechanism remains poorly understood. Here we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the ATP synthase in the inner mitochondrial membrane (IMM) and changes the proton gradient in the mitochondria. This activates NFkB and Jmjd3 expression to remove the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus LPS (M1), both macrophages treated with statins in vitro or isolated from statin-treated mice in vivo, express lower levels pro-inflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.

Regulated adipose tissue-specific expression of human AGPAT2 in lipodystrophic Agpat2-null mice results in regeneration of adipose tissue.

Genetic loss of Agpat2 in humans and mice results in congenital generalized lipodystrophy with near-total loss of adipose tissue and predisposition to develop insulin resistance, diabetes mellitus, hepatic steatosis, and hypertriglyceridemia. The mechanism by which Agpat2 deficiency results in loss of adipose tissue remains unknown. We studied this by re-expressing human AGPAT2 (hAGPAT2) in Agpat2-null mice, regulated by doxycycline. In both sexes of Agpat2-null mice, adipose-tissue-specific re-expression of hAGPAT2 resulted in partial regeneration of both white and brown adipose tissue (but only 30%-50% compared with wild-type mice), which had molecular signatures of adipocytes, including leptin secretion. Furthermore, the stromal vascular fraction cells of regenerated adipose depots differentiated ex vivo only with doxycycline, suggesting the essential role of Agpat2 in adipocyte differentiation. Turning off expression of hAGPAT2 in vivo resulted in total loss of regenerated adipose tissue, clear evidence that Agpat2 is essential for adipocyte differentiation in vivo.

Psychometric evaluation of the Worst Pruritus Numerical Rating Scale (NRS), Atopic Dermatitis Symptom Scale (ADerm-SS), and Atopic Dermatitis Impact Scale (ADerm-IS).

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritus, skin pain, and sleep impacts, which are only reportable by patients themselves. The goal of this research is to evaluate the reliability, validity, and interpretability of the scores from three patient-reported outcome measures within the context of a clinical trial for adolescents and adults with moderate to severe AD. METHODS: Data from a Phase 3 randomized, double-blind, placebo-controlled, multinational clinical trial for individuals 12-75 years of age with moderate to severe AD (AD Up [ClinicalTrials.gov NCT03568318]) were used to assess the reliability, validity, and interpretability of scores on the Worst Pruritus Numerical Rating Scale (NRS) and the Atopic Dermatitis Symptom and Impact Scales (ADerm-SS and ADerm-IS). Analyses were conducted separately for the adult and adolescent subgroups. RESULTS: Of the 882 participants included in the psychometric analyses, the majority were adults (n = 769, 87.2%), male (n = 536, 60.8%), and white (n = 630, 71.4%). Multi-item scores from the ADerm-SS and ADerm-IS had good internal consistency reliability, and most scores demonstrated acceptable test-retest reliability. Scores from the three questionnaires demonstrated adequate validity, exhibiting correlations with other conceptually related outcome assessments and score differences between clinically distinct subgroups. Finally, the score interpretation analyses provide estimates for meaningful within-person change and between-groups difference thresholds that may be useful for future research in adults and adolescents with moderate to severe AD. CONCLUSIONS: These results provide evidence that the scores produced by the Worst Pruritus NRS, ADerm-SS, and ADerm-IS are reliable and construct-valid when completed by adults and adolescents with moderate to severe AD in a clinical trial setting. The results presented here expand upon the previous qualitative evidence of these tools and provide further support for their use in future clinical studies. While results are specific to clinical trials, next steps would be to evaluate the use of these questionnaires in clinical practice. This can provide clinicians and dermatologists a window into the patient's disease experience outside of the clinic, aid in shared decision making, and support a patient-centric approach to management of moderate to severe AD.

Endogenous and Therapeutic 25-Hydroxycholesterols May Worsen Early SARS-CoV-2 Pathogenesis in Mice.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-Hydroxycholesterol (25HC), a product of the activity of cholesterol-25-hydroxylase (CH25H) on cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, 25HC can also amplify inflammation and be converted by CYP7B1 (cytochrome P450 family 7 subfamily B member 1) to 7α,25-dihydroxycholesterol, a lipid with chemoattractant activity, via the G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2)/GPR183 (G protein-coupled receptor 183). Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that although 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 (angiotensin-converting enzyme 2) mouse model in vivo. Treatment with 25HC also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma proinflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points but no change in weight loss. Consistent with these findings, although Ch25h and 25HC were upregulated in the lungs of SARS-CoV-2-infected wild-type mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the ß variant were similar to those in control animals. Taken together, endogenous 25HCs do not significantly regulate early SARS-CoV-2 replication or pathogenesis, and supplemental 25HC may have proinjury rather than therapeutic effects in SARS-CoV-2 pneumonia.

An enteroendocrine-microbial axis in the large intestine controls host metabolism.

Nutrient handling is an essential function of the gastrointestinal tract. Most nutrient absorption occurs in the small intestine and is coordinated by hormone-producing intestinal epithelial cells known as enteroendocrine cells (EECs)1. In contrast, the colon mostly reclaims water and electrolytes, and handles the influx of microbially-derived metabolites, including short chain fatty acids (SCFA)2-4. Hormonal responses of small intestinal EECs have been extensively studied but much less in known about the role of colonic EECs in metabolic regulation. To address this core question, we investigated a mouse model deficient in colonic EECs. We found that colonic EEC deficiency leads to hyperphagia and obesity. Surprisingly, colonic EEC deficiency results in altered microbiota composition and metabolism, which we found through antibiotic treatment and transfer to germ free recipients, to be both necessary and sufficient for the development of obesity. Moreover, studying stool and blood metabolomes, we found that differential glutamate production by intestinal microbiota corresponds to increase appetite due to EEC loss. Finally, we show that colonic glutamate administration can directly increase food intake and activate appetite centers in the central nervous system. These observations shed light on an unanticipated host-microbiota axis in the colon, part of a larger gut-brain axis, that regulates host metabolism and body weight.

Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice.

Hypercholesterolemia and vascular inflammation are key interconnected contributors to the pathogenesis of atherosclerosis. How hypercholesterolemia initiates vascular inflammation is poorly understood. Here we show in male mice that hypercholesterolemia-driven endothelial activation, monocyte recruitment and atherosclerotic lesion formation are promoted by a crosstalk between macrophages and endothelial cells mediated by the cholesterol metabolite 27-hydroxycholesterol (27HC). The pro-atherogenic actions of macrophage-derived 27HC require endothelial estrogen receptor alpha (ERα) and disassociation of the cytoplasmic scaffolding protein septin 11 from ERα, leading to extranuclear ERα- and septin 11-dependent activation of NF-κB. Furthermore, pharmacologic inhibition of cyp27a1, which generates 27HC, affords atheroprotection by reducing endothelial activation and monocyte recruitment. These findings demonstrate cell-to-cell communication by 27HC, and identify a major causal linkage between the hypercholesterolemia and vascular inflammation that partner to promote atherosclerosis. Interventions interrupting this linkage may provide the means to blunt vascular inflammation without impairing host defense to combat the risk of atherosclerotic cardiovascular disease that remains despite lipid-lowering therapies.

Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.