Machine learning methods predict recurrence of pN3b gastric cancer after radical resection.

Background: The incidence of stage pN3b gastric cancer (GC) is low, and the clinical prognosis is poor, with a high rate of postoperative recurrence. Machine learning (ML) methods can predict the recurrence of GC after surgery. However, the prognostic significance for pN3b remains unclear. Therefore, we aimed to predict the recurrence of pN3b through ML models. Methods: This retrospective study included 336 patients with pN3b GC who underwent radical surgery. A 3-fold cross-validation was used to partition the participants into training and test cohorts. Linear combinations of new variable features were constructed using principal component analysis (PCA). Various ML algorithms, including random forest, support vector machine (SVM), logistic regression, multilayer perceptron (MLP), extreme gradient boosting (XGBoost), and Gaussian naive Bayes (GNB), were utilized to establish a recurrence prediction model. Model performance was evaluated using the receiver operating characteristic (ROC) curve and the area under the curve (AUC). Python was used for the analysis of ML algorithms. Results: Nine principal components with a cumulative variance interpretation rate of 90.71% were identified. The output results of the test set showed that random forests had the highest AUC (0.927) for predicting overall recurrence with an accuracy rate of 80.5%. Random forests had the highest AUC (0.940) for predicting regional recurrence with an accuracy of 89.7%. For predicting distant recurrence, random forests had the highest AUC (0.896) with an accuracy of 84.3%. For peritoneal recurrence, random forests had the highest AUC (0.923) with an accuracy of 83.3%. Conclusions: ML can personalize the prediction of postoperative recurrence in patients with GC with stage pN3b.

Development and Validation of a Nomogram for Predicting Tigecycline-Related Coagulopathy: A Retrospective Cohort Study.

Background: Although tigecycline is an effective drug against drug-resistant bacteria, it demonstrated a higher all-cause mortality than comparator antibiotics and a high incidence of coagulation disorders which can be accompanied by severe bleeding. At present, a predictive model for tigecycline-related coagulopathy is not readily available, and the prognostic value of coagulopathy in tigecycline-administered patients has not been elucidated. In this paper, we investigate the association between tigecycline-related coagulopathy and in-hospital mortality to develop a nomogram for the prediction of tigecycline-related coagulopathy. Methods: This retrospective cohort study includes 311 adults prescribed with tigecycline from 2018 to 2020. The primary cohort and validation cohort were constructed by dividing the participants in a ratio of 7:3. The endpoint is tigecycline-related coagulopathy, defined as a condition with no abnormality in coagulation prior to tigecycline application but developed the following symptoms upon prescription: activated partial thromboplastin time (APTT) extended by >10 s than the upper limit of normal (ULN), prothrombin time (PT) prolonged for >3 s than the ULN or reduced serum level of fibrinogen to <2.0 g/L. A predictive nomogram based on logistic regression was subsequently constructed. Results: Tigecycline intake for over 7 days, combined other antibiotics, initial PT, initial fibrinogen and estimated glomerular filtration rate (eGFR), are independent prognostic factors of tigecycline-related coagulopathy. The primary and validation cohort each has an area under the receiver operating characteristic curve (AUC) of 0.792 (0.732-0.851) and 0.730 (0.629-0.832) for nomogram, respectively. Furthermore, the fitted calibration curve illustrated adequate fit of the model, while the decision curve analysis demonstrated good clinical value. Survival curves showed a high mortality rate among patients with tigecycline-related coagulopathy. Conclusion: This nomogram exhibited helpful clinical value in predicting tigecycline-related coagulopathy that could reduce the high mortality rate of patients prescribed with tigecycline.

Sound Target Detection Under Noisy Environment Using Brain-Computer Interface.

As an important means of environmental reconnaissance and regional security protection, sound target detection (STD) has been widely studied in the field of machine learning for a long time. Considering the shortcomings of the robustness and generalization performance of existing methods based on machine learning, we proposed a target detection method by an auditory brain-computer interface (BCI). We designed the experimental paradigm according to the actual application scenarios of STD, recorded the changes in Electroencephalogram (EEG) signals during the process of detecting target sound, and further extracted the features used to decode EEG signals through the analysis of neural representations, including Event-Related Potential (ERP) and Event-Related Spectral Perturbation (ERSP). Experimental results showed that the proposed method achieved good detection performance under noisy environment. As the first study of BCI applied to STD, this study shows the feasibility of this scheme in BCI and can serve as the foundation for future related applications.

A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis.

Phagocytic clearance of dying cells, termed efferocytosis, is essential for maintaining tissue homeostasis, yet our understanding of efferocytosis regulation remains incomplete. Here we perform a FACS-based, genome-wide CRISPR knockout screen in primary mouse macrophages to search for novel regulators of efferocytosis. The results show that Wdfy3 knockout in macrophages specifically impairs uptake, but not binding, of apoptotic cells due to defective actin disassembly. Additionally, WDFY3 interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Mechanistically, while the C-terminus of WDFY3 is sufficient to rescue the impaired degradation induced by Wdfy3 knockout, full-length WDFY3 is required to reconstitute the uptake of apoptotic cells. Finally, WDFY3 is also required for efficient efferocytosis in vivo in mice and in vitro in primary human macrophages. This work thus expands our knowledge of the mechanisms of macrophage efferocytosis, as well as supports genome-wide CRISPR screen as a platform for interrogating complex functional phenotypes in primary macrophages.

Combined metabolomic and transcriptomic profiling approaches reveal the cardiac response to high-fat diet.

The response of vital organs to different types of nutrition or diet is a fundamental question in physiology. We examined the cardiac response to 4 weeks of high-fat diet in mice, measuring cardiac metabolites and mRNA. Metabolomics showed dramatic differences after a high-fat diet, including increases in several acyl-carnitine species. The RNA-seq data showed changes consistent with adaptations to use more fatty acid as substrate and an increase in the antioxidant protein catalase. Changes in mRNA were correlated with changes in protein level for several highly responsive genes. We also found significant sex differences in both metabolomics and RNA-seq datasets, both at baseline and after high fat diet. This work reveals the response of a vital organ to dietary intervention at both metabolomic and transcriptomic levels, which is a fundamental question in physiology. This work also reveals significant sex differences in cardiac metabolites and gene expression.

Mas receptor activation attenuates allergic airway inflammation via inhibiting JNK/CCL2-induced macrophage recruitment.

BACKGROUND: Defective absorption of acute allergic airway inflammation is involved in the initiation and development of chronic asthma. After allergen exposure, there is a rapid recruitment of macrophages around the airways, which promote acute inflammatory responses. The Ang-(1-7)/Mas receptor axis reportedly plays protective roles in various tissue inflammation and remodeling processes in vivo. However, the exact role of Mas receptor and their underlying mechanisms during the pathology of acute allergic airway inflammation remains unclear. OBJECTIVE: We investigated the role of Mas receptor in acute allergic asthma and explored its underlying mechanisms in vitro, aiming to find critical molecules and signal pathways. METHODS: Mas receptor expression was assessed in ovalbumin (OVA)-induced acute asthmatic murine model. Then we estimated the anti-inflammatory role of Mas receptor in vivo and explored expressions of several known inflammatory cytokines as well as phosphorylation levels of MAPK pathways. Mas receptor functions and underlying mechanisms were studied further in the human bronchial epithelial cell line (16HBE). RESULTS: Mas receptor expression decreased in acute allergic airway inflammation. Multiplex immunofluorescence co-localized Mas receptor and EpCAM, indicated that Mas receptor may function in the bronchial epithelium. Activating Mas receptor through AVE0991 significantly alleviated macrophage infiltration in airway inflammation, accompanied with down-regulation of CCL2 and phosphorylation levels of MAPK pathways. Further studies in 16HBE showed that AVE0991 pre-treatment inhibited LPS-induced or anisomycin-induced CCL2 increase and THP-1 macrophages migration via JNK pathways. CONCLUSION: Our findings suggested that Mas receptor activation significantly attenuated CCL2 dependent macrophage recruitments in acute allergic airway inflammation through JNK pathways, which indicated that Mas receptor, CCL2 and phospho-JNK could be potential targets against allergic airway inflammation.

Metformin alleviates allergic airway inflammation and increases Treg cells in obese asthma.

Obesity increases the morbidity and severity of asthma, with poor sensitivity to corticosteroid treatment. Metformin has potential effects on improving asthma airway inflammation. Regulatory T cells (Tregs) play a key role in suppressing the immunoreaction to allergens. We built an obese asthmatic mouse model by administering a high-fat diet (HFD) and ovalbumin (OVA) sensitization, with daily metformin treatment. We measured the body weight and airway inflammatory status by histological analysis, qRT-PCR, and ELISA. The percentage of Tregs was measured by flow cytometry. Obese asthmatic mice displayed more severe airway inflammation and more significant changes in inflammatory cytokines. Metformin reversed the obese situation and alleviated the airway inflammation and remodelling with increased Tregs and related transcript factors. The anti-inflammatory function of metformin may be mediated by increasing Tregs.

Promotor Hypomethylation Mediated Upregulation of miR-23b-3p Targets PTEN to Promote Bronchial Epithelial-Mesenchymal Transition in Chronic Asthma.

Chronic asthma is characterized by airway inflammation and irreversible airway remodeling. Epithelial-mesenchymal transition (EMT) is a typical pathological change of airway remodeling. Our previous research demonstrated miR-23b inhibited airway smooth muscle proliferation while the function of miR-23b-3p has not been reported yet. Besides, miRNA is regulated by many factors, including DNA methylation. The function of miR-23b-3p and whether it is regulated by DNA methylation are worth exploring. Balb/c mice were given OVA sensitization to develop the asthmatic model. Expression of miR-23b-3p and EMT markers were measured by RT-qPCR, WB and immunohistochemistry (IHC). DNA methylation was detected by methylation-specific PCR (MSP) and the MassARRAY System. Asthmatic mice and TGF-ß1-stimulated bronchial epithelial cells (BEAS-2B) showed EMT with increased miR-23b-3p. Overexpression of miR-23b-3p promoted EMT and migration, while inhibition of miR-23b-3p reversed these transitions. DNA methyltransferases were decreased in asthmatic mice. MSP and MassARRAY System detected the promotor of miR-23b showed DNA hypomethylation. DNA methyltransferase inhibitor 5'-AZA-CdZ increased the expression of miR-23b-3p. Meanwhile, PTEN was identified as a target gene of miR-23b-3p. Our results indicated that promotor hypomethylation mediated upregulation of miR-23b-3p targets PTEN to promote EMT in chronic asthma. miR-23b-3p and DNA methylation might be potential therapeutic targets for irreversible airway remodeling.

Two Novel Quassinoid Glycosides with Antiviral Activity from the Samara of Ailanthus altissima.

Phytochemistry investigations on Ailanthus altissima (Mill.) Swingle, a Simaroubaceae plant that is recognized as a traditional herbal medicine, have afforded various natural products, among which C20 quassinoid is the most attractive for their significant and diverse pharmacological and biological activities. Our continuous study has led to the isolation of two novel quassinoid glycosides, named chuglycosides J and K, together with fourteen known lignans from the samara of A. altissima. The new structures were elucidated based on comprehensive spectra data analysis. All of the compounds were evaluated for their anti-tobacco mosaic virus activity, among which chuglycosides J and K exhibited inhibitory effects against the virus multiplication with half maximal inhibitory concentration (IC50) values of 56.21 ± 1.86 and 137.74 ± 3.57 µM, respectively.

Single-Cell Genomics Reveals a Novel Cell State During Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human.

BACKGROUND: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. METHODS: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. RESULTS: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. CONCLUSIONS: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

miR-142-5p and miR-130a-3p regulate pulmonary macrophage polarization and asthma airway remodeling.

Macrophages are key regulators of the development and progression of asthma, facilitating deleterious airway remodeling in affected patients. Immune cell function is tightly regulated by microRNAs (miRNAs), but how these miRNAs impact macrophage-mediated airway remodeling in the context of asthma remains to be determined. In this study, we utilized an ovalbumin (OVA)-based murine model of asthma to evaluate the importance of miRNAs within these macrophages. We found that macrophages from mice that had been sensitized with and exposed to OVA expressed higher levels of M2-like phenotypic markers and exhibited significantly altered expression of both miR-142-5p and miR-130a-3p. When these isolated pulmonary macrophages were cultured in vitro, we determined that transfecting them with miR-142-5p antisense oligonucleotide (ASO) or miR-130a-3p mimics was sufficient to inhibit the ability of interleukin-4 to induce M2 cytokine production. We additionally confirmed the in vivo relevance of these miRNAs in a Ccr2-/- murine model system mimicking asthma. Specifically, we determined that transfecting monocytes with miR-142-5p ASO and/or miR-130a-3p mimics was sufficient to disrupt the ability of these cells to promote airway remodeling. As such, these findings reveal that miR-142-5p and miR-130a-3p dysregulation are important factors governing the polarization of macrophages and associated airway remodeling in OVA-sensitized mice.

Two new compounds from the fruit of Ailanthus altissima.

A new phenolic derivative, 4-hydroxyphenol-1-O-[6-O-(E)-feruloyl-ß-d-glucopyranosyl]-(1→6)-ß-d-glucopyranoside (1), and a new terpenylated coumarin, named altissimacoumarin H (2) were identified from the fruit of Ailanthus altissima (Mill.) Swingle (Simaroubaceae), together with ten known compounds (3-12), including two coumarins and eight phenylpropanoids. Their structures were determined on the basis of chemical method and spectroscopic data. Antiviral effect against Tobacco mosaic virus (TMV) of all the compounds obtained were evaluated using leaf-disc method.

Differentiation of Human-Induced Pluripotent Stem Cells to Macrophages for Disease Modeling and Functional Genomics.

Macrophages play important roles in many diseases. We describe a protocol and the associated resources for the differentiation of human induced pluripotent stem cell-derived macrophages (IPSDM) and their applications in understanding human macrophage physiology and relevant diseases. The protocol uses an embryoid body-based approach with a combination of serum-free condition for hematopoiesis specification, followed by adherent culture with serum and M-CSF for myeloid expansion and macrophage maturation. The protocol produced an almost pure culture of CD45+ /CD18+ macrophages yielding up to 2 × 107 cells per 6-well plate of iPSCs within 24 days, demonstrating high efficiency, purity, and scalability. The IPSDM and monocyte-derived macrophages (HMDM) cultured in the same medium were compared at morphological, functional and transcriptomic levels by RNA-sequencing. IPSDM and HMDM showed broadly similar profiles of coding transcriptome, alternative splicing events, and long noncoding RNAs, with advantages and successful applications in disease modeling using patients-derived and CRISPR-edited iPSC lines. © 2018 by John Wiley & Sons, Inc.

Tissue-Specific Differential Expression of Novel Genes and Long Intergenic Noncoding RNAs in Humans With Extreme Response to Evoked Endotoxemia.

BACKGROUND: Cytokine responses to activation of innate immunity differ between individuals, yet the genomic and tissue-specific transcriptomic determinants of inflammatory responsiveness are not well understood. We hypothesized that tissue-specific mRNA and long intergenic noncoding RNA (lincRNA) induction differs between individuals with divergent evoked inflammatory responses. METHODS: In the GENE Study (Genetics of Evoked Response to Niacin and Endotoxemia), we performed an inpatient endotoxin challenge (1 ng/kg lipopolysaccharide [LPS]) in healthy humans. We selected individuals in the top (high responders) and bottom (low responders) extremes of inflammatory responses and applied RNA sequencing to CD14 monocytes (N=15) and adipose tissue (N=25) before and after LPS administration. RESULTS: Although only a small number of genes were differentially expressed at baseline, there were clear differences in the magnitude of the transcriptional response post-LPS between high and low responders, with a far greater number of genes differentially expressed by endotoxemia in high responders. Furthermore, tissue responses differed during inflammation, and we found a number of tissue-specific differentially expressed lincRNAs post-LPS, which we validated. Relative to nondifferentially expressed lincRNAs, differentially expressed lincRNAs were equally likely to be nonconserved as conserved between human and mouse, indicating that conservation is not a predictor of lincRNAs associated with human inflammatory pathophysiology. Differentially expressed genes also were enriched for signals with inflammatory and cardiometabolic disease in published genome-wide association studies. CTB-41I6.2 ( AC002091.1), a nonconserved human-specific lincRNA, is one of the top lincRNAs regulated by endotoxemia in monocytes, but not in adipose tissue. Knockdown experiments in THP-1 monocytes suggest that this lincRNA enhances LPS-induced interleukin 6 ( IL6) expression in monocytes, and we now refer to this as monocyte LPS-induced lincRNA regulator of IL6 ( MOLRIL6). CONCLUSIONS: We highlight mRNAs and lincRNAs that represent novel candidates for modulation of innate immune and metabolic responses in humans. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT00953667.

TGF-ß1-Induced Airway Smooth Muscle Cell Proliferation Involves TRPM7-Dependent Calcium Influx via TGFßR/SMAD3.

BACKGROUND: TRPM7 in mast cells plays a key role in asthma. However, the effect of TRPM7 on TGF-ß1-induced airway smooth muscle cell (ASMC) proliferation remains unclear. Therefore, we designed this study to explore whether TRPM7 is involved in TGF-ß1-induced ASMC proliferation. METHODS: An asthmatic mouse model was established, and the expressions of TGF-ß1 and TRPM7 in mice lungs were detected using enzyme-linked immunosorbent assay (ELISA) and western blotting. In addition, murine ASMCs were cultured and stimulated with TGF-ß1. Possible TGF-ß1 and TRPM7 interactions were examined using RT-PCR and western blotting analyses with ASMCs. The effect of TRPM7 knockdown on ASMC calcium influx was assessed by the fluorescent indicator indo-1. MTT and flow cytometry were applied to evaluate the effects of TRPM7 knockdown on ASMC proliferation and apoptosis. RESULTS: TGF-ß1 elevated the expression of TRPM7 via TGFßR/SMAD3. Knocking down TRPM7 decreased the intracellular Ca2+ concentration and cellular proliferation of ASMCs, although apoptosis remained unaffected. CONCLUSIONS: Our initial findings suggest that TRPM7 inhibition may prevent asthma-induced airway remodeling.

Anti-Tobacco Mosaic Virus Quassinoids from Ailanthus altissima (Mill.) Swingle.

Quassinoids are bitter constituents characteristic of the family Simaroubaceae. A total of 18 C20 quassinoids, including nine new quassinoid glycosides, named chuglycosides A-I (1-6 and 8-10), were identified from the samara of Ailanthus altissima (Mill.) Swingle. All of the quassinoids showed potent anti-tobacco mosaic virus (TMV) activity. A preliminary structure-anti-TMV activity relationship of quassinoids was discussed. The effects of three quassinoids, including chaparrinone (12), glaucarubinone (15), and ailanthone (16), on the accumulation of TMV coat protein (CP) were studied by western blot analysis. Ailanthone (16) was further investigated for its influence on TMV spread in the Nicotiana benthamiana plant.

Phenylpropionamides, Piperidine, and Phenolic Derivatives from the Fruit of Ailanthus altissima.

Four novel compounds-two phenylpropionamides, one piperidine, and one phenolic derivatives-were isolated and identified from the fruit of a medicinal plant, Ailanthus altissima (Mill.) Swingle (Simaroubaceae), together with one known phenylpropionamide, 13 known phenols, and 10 flavonoids. The structures of the new compounds were elucidated as 2-hydroxy-N-[(2-O-ß-d-glucopyranosyl)phenyl]propionamide (1), 2-hydroxy-N-[(2-O-ß-d-glucopyranosyl-(1→6)-ß-d-glucopyranosyl)phenyl]propionamide (2), 2ß-carboxyl-piperidine-4ß-acetic acid methyl ester (4), and 4-hydroxyphenyl-1-O-[6-(hydrogen-3-hydroxy-3-methylpentanedioate)]-ß-d-glucopyranoside (5) based on spectroscopic analysis. All the isolated compounds were evaluated for their inhibitory activity against Tobacco mosaic virus (TMV) using the leaf-disc method. Among the compounds isolated, arbutin (6), ß-d-glucopyranosyl-(1→6)-arbutin (7), 4-methoxyphenylacetic acid (10), and corilagin (18) showed moderate inhibition against TMV with IC50 values of 0.49, 0.51, 0.27, and 0.45 mM, respectively.

Deep RNA Sequencing Uncovers a Repertoire of Human Macrophage Long Intergenic Noncoding RNAs Modulated by Macrophage Activation and Associated With Cardiometabolic Diseases.

BACKGROUND: Sustained and dysfunctional macrophage activation promotes inflammatory cardiometabolic disorders, but the role of long intergenic noncoding RNA (lincRNA) in human macrophage activation and cardiometabolic disorders is poorly defined. Through transcriptomics, bioinformatics, and selective functional studies, we sought to elucidate the lincRNA landscape of human macrophages. METHODS AND RESULTS: We used deep RNA sequencing to assemble the lincRNA transcriptome of human monocyte-derived macrophages at rest and following stimulation with lipopolysaccharide and IFN-γ (interferon γ) for M1 activation and IL-4 (interleukin 4) for M2 activation. Through de novo assembly, we identified 2766 macrophage lincRNAs, including 861 that were previously unannotated. The majority (≈85%) was nonsyntenic or was syntenic but not annotated as expressed in mouse. Many macrophage lincRNAs demonstrated tissue-enriched transcription patterns (21.5%) and enhancer-like chromatin signatures (60.9%). Macrophage activation, particularly to the M1 phenotype, markedly altered the lincRNA expression profiles, revealing 96 lincRNAs differentially expressed, suggesting potential roles in regulating macrophage inflammatory functions. A subset of lincRNAs overlapped genomewide association study loci for cardiometabolic disorders. MacORIS (macrophage-enriched obesity-associated lincRNA serving as a repressor of IFN-γ signaling), a macrophage-enriched lincRNA not expressed in mouse macrophages, harbors variants associated with central obesity. Knockdown of MacORIS, which is located in the cytoplasm, enhanced IFN-γ-induced JAK2 (Janus kinase 2) and STAT1 (signal transducer and activator of transcription 1) phosphorylation in THP-1 macrophages, suggesting a potential role as a repressor of IFN-γ signaling. Induced pluripotent stem cell-derived macrophages recapitulated the lincRNA transcriptome of human monocyte-derived macrophages and provided a high-fidelity model with which to study lincRNAs in human macrophage biology, particularly those not conserved in mouse. CONCLUSIONS: High-resolution transcriptomics identified lincRNAs that form part of the coordinated response during macrophage activation, including specific macrophage lincRNAs associated with human cardiometabolic disorders that modulate macrophage inflammatory functions.

CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

OBJECTIVE: To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. APPROACH AND RESULTS: We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA (LIPA-/-) had barely detectable LAL enzymatic activity. Control and LIPA-/- IPSDM were loaded with [3H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [3H]-cholesterol to apolipoprotein A-I was abolished in LIPA-/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [3H]-cholesterol-labeled AcLDL, [3H]-cholesterol efflux was, however, not different between control and LIPA-/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA-/- IPSDM. In nonlipid loaded state, LIPA-/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA-/- IPSDM. LIPA-/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B, IL6, and CCL5. CONCLUSIONS: LIPA-/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human macrophages.