Primary Cultures Of Human Cells Research Articles

Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Liquid application dosing alters the physiology of air-liquid interface (ALI) primary human bronchial epithelial cell/lung fibroblast co-cultures and in vitro testing relevant endpoints.

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by "liquid application," involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis.

Introduction: Autologous transplantation of spermatogonial stem cells (SSCs) isolated from cryopreserved testicular biopsies obtained before oncological treatment could restore fertility in male childhood cancer survivors. There is a clear necessity for in vitro propagation of the limited SSCs from the testicular biopsy prior to transplantation due to limited numbers of spermatogonia in a cryopreserved testicular biopsy. Still, there is no consensus regarding their optimal culture method. Methods: We performed a systematic review and meta-analysis of studies reporting primary testicular cell cultures of human and non-human primate origin through use of Pubmed, EMBASE, and Web of Science core collection databases. Of 760 records, we included 42 articles for qualitative and quantitative analysis. To quantify in vitro spermatogonial propagation, spermatogonial colony doubling time (CDT) was calculated, which measures the increase in the number of spermatogonial colonies over time. A generalized linear mixed model analysis was used to assess the statistical effect of various culture conditions on CDT. Results: Our analysis indicates decreased CDTs, indicating faster spermatogonial propagation in cultures with a low culture temperature (32°C); with use of non-cellular matrices; use of StemPro-34 medium instead of DMEM; use of Knockout Serum Replacement; and when omitting additional growth factors in the culture medium. Discussion: The use of various methods and markers to detect the presence of spermatogonia within the reported cultures could result in detection bias, thereby potentially influencing comparability between studies. However, through use of CDT in the quantitative analysis this bias was reduced. Our results provide insight into critical culture conditions to further optimize human spermatogonial propagation in vitro, and effectively propagate and utilize these cells in a future fertility restoration therapy and restore hope of biological fatherhood for childhood cancer survivors.

Multi‐omic study of a cellular model of the retinal pigment epithelium producing extracellular deposits

Aims/Purpose: To study transcriptomic and metabolomic changes during a 6‐month follow‐up of an in vitro model of the retinal pigment epithelium (RPE) producing extracellular deposits.Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts for 6 months and harvested at 4, 12, 17 and 25 weeks. Progressive accumulation of extracellular material was determined by apolipoprotein E (APOE) and hydroxyapatite (HAP) localization. Transcriptome was analysed by RNA sequencing (RNAseq) and differential expression analysis was conducted using the platform Dr. Tom. Untargeted metabolomics was carried out by high‐resolution mass spectrometry (LC‐IM‐QTOF) and bioinformatic analysis was performed using Mass Professional Profiler software.Results: RPE cells formed a fully differentiated monolayer at 4 weeks in culture, but tight junctions and electrical resistance decreased from 12 weeks onwards, coinciding with the formation of extracellular deposits containing APOE and HAP. RNAseq showed alterations of cell migration, adhesion and differentiation, and oxidation–reduction, lipid metabolic and apoptotic processes during RPE ageing (4 vs 25 weeks, Q‐value < 0.001). Untargeted metabolomics provided 350 non‐redundant compounds, and principal component analysis showed a clear separation between time points and good reliability within each group (55% of cumulative variance). Moderated t‐test (Benjamin‐Hochberg correction) provided 103 compounds differentially altered during RPE follow‐up (p‐value < 0.001, fold‐change > 2), belonging to trans‐sulfuration, one carbon metabolism, glutathione metabolism, Kennedy pathway for sphingolipids and cysteine and methionine catabolism.Conclusions: Time‐dependent changes were observed in the cell culture model, including progressive sub‐RPE accumulation of APOE and HAP, and alterations in cell adhesion and migration, lipid metabolism and sphingolipids pathway contributing to photoreceptor protection and calcium mobilization.

Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Study of the metallome and transcriptome of a degenerating cellular model of the retinal pigment epithelium

Aims/Purpose: To study the changes on the metallome and transcriptome of a cellular model of the retinal pigment epithelium (RPE) during its in vitro degeneration after 4 months in culture.Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts and cultured for 4 months. Cell pigmentation and barrier function was followed up using optical microscopy and measurement of the transepithelial electrical resistance (TEER). Samples were collected at 21, 91 and 133 days in culture and analysed for gene expression, metal homeostasis of Ca, Na, P, Mg and P, and extracellular deposit formation through RNA sequencing (RNA‐Seq), elemental mass spectrometry (ICP‐MS) and immunocytochemistry assays, respectively.Results: RPE cells formed a differentiated monolayer at 21 days in culture, expressing specific genes and proteins of RPE and giving increasing TEER values. However, after 133 days in culture, loss of tight junction proteins (i.e. CLDN19 and BEST1) and electrical resistance was seen, together with loss of pigmentation and increase of apolipoprotein E deposition in the extracellular space. Gene expression analysis showed an enrichment on apoptotic processes, biomolecule phosphorylation, and signs of epithelial‐to‐mesenchymal transition (EMT). Degeneration of RPE cells concurred with an increase of Na and P content in the cytosol and decrease of Mg, while a decrease of Mg, P and Cu was determined in the insoluble fraction of cells. Time‐dependent changes of metals were accompanied with the alteration of metal transporters and antioxidant enzymes using metals as cofactors.Conclusions: Degeneration of RPE cells in culture was characterized by depigmentation of cells, improper barrier function, downregulation of antioxidant protection, loss of epithelial phenotype, and changes on the levels of Na, P, Mg and Cu, as well as of the expression of their homeostatic proteins.

Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells.

Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD.

Mitochondrial uncoupling proteins regulate the metabolic function of human Sertoli cells.

Mitochondrial uncoupling proteins (UCPs) regulate mitochondrial activity and reactive oxygen species production through the transport of protons and metabolites. This study identified the expression of UCPs in human Sertoli cells, which proved to be modulators of their mitochondrial activity. Mitochondrial uncoupling proteins (UCPs) are mitochondrial channels responsible for the transport of protons and small molecular substrates across the inner mitochondrial membrane. Altered UCP expression or function is commonly associated with mitochondrial dysfunction and increased oxidative stress, which are both known causes of male infertility. However, UCP expression and function in the human testis remain to be characterized. This study aimed to assess the UCP homologs (UCP1-6) expression and function in primary cultures of human Sertoli cells (hSCs). We identified the mRNA expression of all UCP homologs (UCP1-6) and protein expression of UCP1, UCP2, and UCP3 in hSCs. UCP inhibition by genipin for 24 h decreased hSCs proliferation without causing cytotoxicity (n = 6). Surprisingly, the prolonged UCP inhibition for 24 h decreased mitochondrial membrane potential, oxygen consumption rate (OCR), and endogenous reactive oxygen species (ROS) production. The metabolism of hSCs was also affected as UCP inhibition shifted their metabolism toward an increased pyruvate consumption. Taken together, these findings demonstrate that UCPs play a role as regulators of the mitochondrial function in hSCs, emphasizing their potential as targets in the study of male (in)fertility.

Reciprocal mutations of lung-tropic AAV capsids lead to improved transduction properties.

Considerable effort has been devoted to developing adeno-associated virus (AAV)-based vectors for gene therapy in cystic fibrosis (CF). As a result of directed evolution and capsid shuffling technology, AAV capsids are available with widespread tropism for airway epithelial cells. For example, AAV2.5T and AAV6.2 are two evolved capsids with improved airway epithelial cell transduction properties over their parental serotypes. However, limited research has been focused on identifying their specific cellular tropism. Restoring cystic fibrosis transmembrane conductance regulator (CFTR) expression in surface columnar epithelial cells is necessary for the correction of the CF airway phenotype. Basal cells are a progenitor population of the conducting airways responsible for replenishing surface epithelial cells (including secretory cells and ionocytes), making correction of this cell population vital for a long-lived gene therapy strategy. In this study, we investigate the tropism of AAV capsids for three cell types in primary cultures of well-differentiated human airway epithelial (HAE) cells and primary human airway basal cells. We observed that AAV2.5T transduced surface epithelial cells better than AAV6.2, while AAV6.2 transduced airway basal cells better than AAV2.5T. We also investigated a recently developed capsid, AAV6.2FF, which has two surface tyrosines converted to phenylalanines. Next, we incorporated reciprocal mutations to create AAV capsids with further improved surface and basal cell transduction characteristics. Lastly, we successfully employed a split-intein approach using AAV to deliver an adenine base editor (ABE) to repair the CFTR R553X mutation. Our results suggest that rational incorporation of AAV capsid mutations improves AAV transduction of the airway surface and progenitor cells and may ultimately lead to improved pulmonary function in people with CF.

An effective method for culturing functional human corneal endothelial cells using a xenogeneic free culture medium

Endothelial dysfunction is a leading cause of corneal blindness in developed countries and the only available treatment is the endothelial transplantation. However, the limited availability of suitable donors remains a significant challenge, driving the exploration of alternative regenerative therapies. Advanced Therapy Medicinal Products show promise but must adhere to strict regulations that prohibit the use of animal-derived substances. This study investigates a novel culture methodology using Plasma Rich in Growth Factors (PRGF) as the only source of growth factors for primary cultures of human corneal endothelial cells (CECs). CECs were obtained from discarded corneas or endothelial rings and cultured in two different media: one supplemented with xenogeneic factors and other xenogeneic-free, using PRGF. Comprehensive characterization through immunofluorescence, morphological analyses, trans-endothelial electrical resistance measurements, RNA-seq, and qPCR was conducted on the two groups. Results demonstrate that CECs cultured in the xenogeneic-free medium exhibit comparable gene expression, morphology, and functionality to those cultured in the xenogeneic medium. Notably, PRGF-expanded CECs share 46.9% of the gene expression profile with native endothelium and express all studied endothelial markers. In conclusion, PRGF provides an effective source of xenogeneic-free growth factors for the culture of CECs from discarded corneal tissue. Further studies will be necessary to demonstrate the applicability of these cultures to cell therapies that make clinical translation possible.

Abstract 18413: Therapeutic Genome Editing of Endothelial FABP4 by Endothelium-Targeted Nanoparticle Delivery of the CRISPR/Cas9 System Inhibits Pulmonary Arterial Hypertension in Mice and Rats

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling. The molecular mechanisms of obliterative pulmonary vascular remodeling remain elusive. Hypothesis: Therapeutic genome editing of endothelial Fabp4 will knockout FABP4 expression selectively in endothelial cells and inhibit PAH in mice and rats. Methods: Employing the newly developed endothelial cell (EC)-targeted nanoparticle delivery technology, plasmid DNA expressing CRISPR/Cas9 controlled by Cdh5 promoter and Fabp4 -specific guide RNAs driven by U6 promoter was administered i.v. to adult SD rats and Egln1 Tie2Cre mice which exhibit severe PAH and obliterative pulmonary vascular remodeling. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, right ventricular systolic pressure (RVSP), ratio of right ventricular versus left ventricular plus septum (RV/LV+S) and pulmonary vascular remodeling were determined. Pharmacological FABP4 inhibitor was also employed to treat MCT rats as a comparison. Similarly, the pulmonary hypertensive phenotype in genome-edited Egln1 Tie2Cre mice was also characterized. Results: Nanoparticle delivery of the all-in-one CRISPR rCdh5 plasmid DNA knockout Fabp4 selectively in endothelial cells in adult rats and Egln1 Tie2Cre mice leading to reduced RVSP, RV hypertrophy and pulmonary vascular remodeling in MCT rats and also in Egln1 Tie2Cre mice. Obliterative pulmonary vascular remodeling seen in Egln1 Tie2Cre mice was inhibited and the mortality rate in endothelial Fabp4-deficient Egln1 Tie2Cre mice was reduced. Treatment of FABP4 inhibitor also inhibits PAH in MCT rats. We also observed a marked increase of FABP4 expression in endothelial cells of lung samples of IPAH patients. Overexpression of FABP4 in primary cultures of human lung microvascular endothelial cells resulted in a 70% increase of cell proliferation. Conclusions: These findings demonstrate endothelial FABP4 is a promising target for PAH therapy. EC-targeted nanoparticle delivery of the genome editing system may represent an effective and safer gene therapy approach for PAH treatment.

Primary Human Nasal Epithelial Cell Culture.

Primary human nasal epithelial cell (HNEC) cultures serve as an excellent model in studying physiology, disease, and therapy related to the respiratory tract. The current chapter details protocols from sample collection to culturing HNEC under air-liquid interface (ALI) conditions to generate well-differentiated, functional airway cultures including displaying beating motile cilia.

Transcriptional landscape of human trophoblast cells treated with calcitriol and TGF-β1

Calcitriol and transforming growth factor beta 1 (TGF-β1) are unrelated molecules that regulate biological processes according to the genetic target, cell type, and context. Several studies have shown independent effects of calcitriol and TGF-βs on the placenta, but there is no information regarding the impact of their combination on these cells. Therefore, this study analyzed the effects of calcitriol, TGF-β1, and their combination in primary cultures of human trophoblast cells using a whole genome expression microarray. Data analysis revealed a set of differentially expressed genes induced by each treatment. Enrichment pathway analysis identified modulatory effects of calcitriol on genes related to metabolic processes such as vitamin D, steroid, and fat-soluble vitamins as well as antimicrobial and immune responses. In relation to TGF-β1, the analysis showed a few differentially expressed genes that were mainly associated with the neutrophil immune response. Lastly, the analysis revealed that the combination of calcitriol and TGF-β1 up-regulated genes involving both immunologic processes and the biosynthesis of unsaturated fatty acids, eicosanoids, and lipoxins, among others. In contrast, pathways down-regulated by the combination were mostly associated with the catabolic process of acylglycerols and peptides, PPAR signaling pathway, cellular response to low-density lipoprotein stimulus, renin angiotensin system and digestion, mobilization and transport of lipids. Consistent with these results, the combined treatment on human trophoblast cells induced the accumulation of intracellular neutral lipid droplets and stimulated both gene and protein expression of 15-hydroxyprostaglandin dehydrogenase. In conclusion, the results revealed that differentially expressed genes induced by the combination modified the transcriptional landscape compared to each treatment alone, mainly altering the storage, activity and metabolism of lipids, which might have an impact on placental development.

Muscarinic receptor agonist-induced βPix binding to β-catenin promotes colon neoplasia

M3 muscarinic receptors (M3R) modulate β-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, βPix, binds to β-catenin in colon cancer cells, augmenting β-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of βPix and β-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of βPix with β-catenin. βPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing βPix dose-dependently augmented β-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of βPix. Expression levels of β-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from βPix-deficient conditional knockout mice. Targeting the M3R/βPix/β-catenin axis may have therapeutic potential.

SAT263 Osteocalcin Promotes Cortisol Production In Human Adrenal Cells

Abstract Disclosure: R. Scheske: None. D. van Rooyen: None. W.E. Rainey: None. Background: Osteocalcin (OCN) is a peptide exclusively produced by osteoblasts in bone. In an uncarboxylated form, OCN has been shown to function as a hormone affecting physiological processes such as reproduction and energy metabolism in mice and humans. Murine studies have shown that uncarboxylated OCN upregulates fetal mouse glucocorticoid production through a unique regulatory axis between osteoblasts and adrenal cortex cells. The increase in adrenal steroid production has been attributed to OCN heightening adrenocorticotropic hormone (ACTH). The influence of adrenal glucocorticoids on glucose metabolism and bone composition is well studied but the effect of OCN on human adrenal steroidogenesis remains unknown. Herein, we hypothesized OCN would stimulate cortisol production in human adrenal cells. Methods: OCN’s effect on adrenal glucocorticoid production was investigated using primary cultures of human adrenal cells (PAC). Cells were treated for 24h with 1000 nM OCN with/without cAMP/protein kinase A (PKA) signaling pathway agonists (ACTH, forskolin, 8-bromo cAMP (8Br-cAMP), and dibutyryl cyclic AMP (dbcAMP)). Cortisol levels were quantified using an enzyme-linked immunosorbent assay. RNA was isolated and assessed by quantitative real-time PCR (RT-qPCR) to identify changes in transcript levels of enzymes catalyzing glucocorticoid production: cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase/17,20-lyase (CYP17A1), and 11β-hydroxylase (CYP11B1). Results: Cortisol production in PAC were significantly induced by all tested agonists; ACTH (22-fold), forskolin (27-fold), 8Br-cAMP (26-fold), and dbcAMP (30-fold) relative to basal. Although OCN, alone, did not significantly increase cortisol in this cell model, co-treatment with ACTH increased cortisol levels 1.4-fold (40.1 ng/mL; p<0.0001) compared to ACTH (28.2 ng/mL). Similarly, co-treatment with forskolin or 8Br-cAMP increased cortisol, relative to the respective agonists, by 1.3-fold (p<0.001) and dbcAMP by 1.2-fold (p<0.01). RT-qPCR analysis showed no changes in CYP11A1 or CYP17A1 transcript levels in co-treatments with OCN and ACTH relative to ACTH. However, CYP11B1 mRNA levels, relative to the respective controls, were increased in samples co-treated with OCN and ACTH/-agonist. OCN co-treatments with ACTH, forskolin, 8Br-cAMP and dbcAMP increased CYP11B1 levels by 1.8-, 1.4-, 1.3-, and 1.2-fold, respectively. Conclusion: Data presented in this study show stimulatory effect of OCN on human adrenal cells. OCN, in the presence of ACTH and other PKA agonists, increased cortisol production via upregulating CYP11B1 transcript levels. Defining the mechanism and receptor interaction through which OCN functions in human adrenal cells remain to be determined. Presentation: Saturday, June 17, 2023

304 Effect of culture conditions on CFTR activity in primary human nasal epithelial cell cultures

304 Effect of culture conditions on CFTR activity in primary human nasal epithelial cell cultures

The Influenza B Virus Victoria and Yamagata Lineages Display Distinct Cell Tropism and Infection-Induced Host Gene Expression in Human Nasal Epithelial Cell Cultures.

Understanding Influenza B virus infections is of critical importance in our efforts to control severe influenza and influenza-related diseases. Until 2020, two genetic lineages of influenza B virus-Yamagata and Victoria-circulated in the population. These lineages are antigenically distinct, but the differences in virus replication or the induction of host cell responses after infection have not been carefully studied. Recent IBV clinical isolates of both lineages were obtained from influenza surveillance efforts of the Johns Hopkins Center of Excellence in Influenza Research and Response and characterized in vitro. B/Victoria and B/Yamagata clinical isolates were recognized less efficiently by serum from influenza-vaccinated individuals in comparison to the vaccine strains. B/Victoria lineages formed smaller plaques on MDCK cells compared to B/Yamagata, but infectious virus production in primary human nasal epithelial cell (hNEC) cultures showed no differences. While ciliated epithelial cells were the dominant cell type infected by both lineages, B/Victoria lineages had a slight preference for MUC5AC-positive cells, and B/Yamagata lineages infected more basal cells. Finally, while both lineages induced a strong interferon response 48 h after infection of hNEC cultures, the B/Victoria lineages showed a much stronger induction of interferon-related signaling pathways compared to B/Yamagata. This demonstrates that the two influenza B virus lineages differ not only in their antigenic structure but also in their ability to induce host innate immune responses.

Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells.

Amplification of measles virus (MeV) in human airway epithelia may contribute to its extremely high contagious nature. We use well-differentiated primary cultures of human airway epithelial cells (HAE) to model ex vivo how MeV spreads in human airways. In HAE, MeV spreads cell-to-cell for 3-5 days, but then, infectious center growth is arrested. What stops MeV spread in HAE is not understood, but interferon (IFN) is known to slow MeV spread in other in vitro and in vivo models. Here, we assessed the role of type I and type III IFN in arresting MeV spread in HAE. The addition of IFN-β or IFN-λ1 to the medium of infected HAE slowed MeV infectious center growth, but when IFN receptor signaling was blocked, infectious center size was not affected. In contrast, blocking type-I IFN receptor signaling enhanced respiratory syncytial virus spread. HAE were also infected with MeV mutants defective for the V protein. The V protein has been demonstrated to interact with both MDA5 and STAT2 to inhibit activation of innate immunity; however, innate immune reactions were unexpectedly muted against the V-defective MeV in HAE. Minimal innate immunity activation was confirmed by deep sequencing, quantitative RT-PCR, and single-cell RNA-seq analyses of the transcription of IFN and IFN-stimulated genes. We conclude that in HAE, IFN-signaling can contribute to slowing infectious center growth; however, IFN-independent processes are most important for limiting cell-to-cell spread. IMPORTANCE Fundamental biological questions remain about the highly contagious measles virus (MeV). MeV amplifies within airway epithelial cells before spreading to the next host. This final step likely contributes to the ability of MeV to spread host-to-host. Over the course of 3-5 days post-infection of airway epithelial cells, MeV spreads directly cell-to-cell and forms infectious centers. Infectious center formation is unique to MeV. In this study, we show that interferon (IFN) signaling does not explain why MeV cell-to-cell spread is ultimately impeded within the cell layer. The ability of MeV to spread cell-to-cell in airway cells without appreciable IFN induction may contribute to its highly contagious nature. This study contributes to the understanding of a significant global health concern by demonstrating that infectious center formation occurs independent of the simplest explanation for limiting viral transmission within a host.

P12.08.A CELL-EXTRINSIC D-2-HYDROXYGLUTARATE PROMOTES HYPERMETHYLATION AT LINEAGE-SPECIFIC REGULATORY REGIONS TO PREVENT MICROGLIAL ACTIVATION IN IDH-MUTANT GLIOMAS

Abstract BACKGROUND Tumor-associated macrophages and microglia (TAMs) are highly abundant myeloid cells in diffuse gliomas. Their composition and transcriptional programs differ according to the IDH mutation status of glioma cells. The underlying mechanisms remain until now little known. MATERIAL AND METHODS We compared bulk DNA methylome (Methylation EPIC array) and transcriptome of TAMs (CD11b+ purified by magnetic-activated cell sorting) from 25 IDH-mutant and 11 IDH-wildtype gliomas. The contamination of the CD11b+ fraction by tumor cells was determined by the detection of IDH and/or TERT mutations using droplet digital PCR. Only samples with a purity of at least 95% were used for analyses. To determine experimentally direct effects of D-2-hydroxyglutarate (D-2-HG), which is the oncometabolite produced and realeased into the microenvironment by IDH-mutated glioma cells, we used primary cultures of human microglial cells obtained from glioma surgery or epilepsy surgery. We exposed microglia or not to (D-2-HG) during 14 days, and analyzed their DNA methylome and transcriptome. RESULTS TAMs from IDH-mutant gliomas compared with TAMs from IDH-wildtype gliomas exhibit decreased expression of pro-inflammatory pathways (mainly IFN-γ, IFN-α, and TNF-α signaling) and genes related to G2-M checkpoint, glycolysis and Epithelial-Mesenchymal Transition (EMT). We uncovered that TAMs from IDH-mutant gliomas exhibit significant hypermethylation at promoters and enhancers linked to decreased expression of genes involved in inflammatory responses. Notably, these hypermethylated regions were enriched for binding motifs of transcription factors controlling an environment-dependent transcriptional network in microglial cells (such as PU.1, AP-1, C/EBP and RUNX). These findings were confirmed by comparing methylome data of TAMs from IDH-mutant gliomas and methylome of microglia from normal brains available in the literature. A 14 days exposition to D-2-HG of human microglial cells in vitro partially recapitulates the observed methylation changes. Transcriptome analyses of these cells show that this oncometabolite dampens the IFN-α/γ pathways and attenuates the response to LPS while inducing a metabolic shift toward oxidative phosphorylation. CONCLUSION Our findings establish a link between an extrinsic cell-effect of D-2-HG and the hypermethylation of DNA of microglial cells at regulatory regions in IDH-mutant gliomas. Our observations support the concept that oncometabolites may disrupt directly the function of immune cells of the tumor microenvironment.

Comparative polar and lipid plasma metabolomics differentiate KSHV infection and disease states

BackgroundKaposi sarcoma (KS) is a neoplastic disease etiologically associated with infection by the Kaposi sarcoma-associated herpesvirus (KSHV). KS manifests primarily as cutaneous lesions in individuals due to either age (classical KS), HIV infection (epidemic KS), or tissue rejection preventatives in transplantation (iatrogenic KS) but can also occur in individuals, predominantly in sub-Saharan Africa (SSA), lacking any obvious immune suppression (endemic KS). The high endemicity of KSHV and human immunodeficiency virus-1 (HIV) co-infection in Africa results in KS being one of the top 5 cancers there. As with most viral cancers, infection with KSHV alone is insufficient to induce tumorigenesis. Indeed, KSHV infection of primary human endothelial cell cultures, even at high levels, is rarely associated with long-term culture, transformation, or growth deregulation, yet infection in vivo is sustained for life. Investigations of immune mediators that distinguish KSHV infection, KSHV/HIV co-infection, and symptomatic KS disease have yet to reveal consistent correlates of protection against or progression to KS. In addition to viral infection, it is plausible that pathogenesis also requires an immunological and metabolic environment permissive to the abnormal endothelial cell growth evident in KS tumors. In this study, we explored whether plasma metabolomes could differentiate asymptomatic KSHV-infected individuals with or without HIV co-infection and symptomatic KS from each other.MethodsTo investigate how metabolic changes may correlate with co-infections and tumorigenesis, plasma samples derived from KSHV seropositive sub-Saharan African subjects in three groups, (A) asymptomatic (lacking neoplastic disease) with KSHV infection only, (B) asymptomatic co-infected with KSHV and HIV, and (C) symptomatic with clinically diagnosed KS, were subjected to analysis of lipid and polar metabolite profilesResultsPolar and nonpolar plasma metabolic differentials were evident in both comparisons. Integration of the metabolic findings with our previously reported KS transcriptomics data suggests dysregulation of amino acid/urea cycle and purine metabolic pathways, in concert with viral infection in KS disease progression.ConclusionsThis study is, to our knowledge, the first to report human plasma metabolic differentials between in vivo KSHV infection and co-infection with HIV, as well as differentials between co-infection and epidemic KS.