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Endothelial dysfunction plays a central role in the severity of COVID-19, since the respiratory, thrombotic and myocardial complications of the disease are closely linked to vascular endothelial damage. To address this issue, we evaluate here the effect of conditioned media from spike S1-activated macrophages (CM_S1) on the proliferation of human umbilical endothelial cells (HUVECs), focusing on the specific role of interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Results obtained demonstrate that the incubation with CM_S1 for 72 h hinders endothelial cell proliferation and induces signs of cytotoxicity. Comparable results are obtained upon exposure to IFN-γ + TNF-α, which are thus postulated to play a pivotal role in the effects observed. These events are associated with an increase in p21 protein and a decrease in Rb phosphorylation, as well as with the activation of IRF-1 and NF-kB transcription factors. Overall, these findings further sustain the pivotal role of a hypersecretion of inflammatory cytokines as a trigger for endothelial activation and injury in the immune-mediated effects of COVID-19. [ABSTRACT FROM AUTHOR]

Abstract Background In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of SLC7A7/y+LAT1 impair system y+L transport activity for cationic amino acids. A severe complication of LPI is a form of Pulmonary Alveolar Proteinosis (PAP), in which alveolar spaces are filled with lipoproteinaceous material because of the impaired surfactant clearance by resident macrophages. The pathogenesis of LPI-associated PAP remains still obscure. The present study investigates for the first time the expression and function of y+LAT1 in monocytes and macrophages isolated from a patient affected by LPI-associated PAP. A comparison with mesenchymal cells from the same subject has been also performed. Methods Monocytes from peripheral blood were isolated from a 21-year-old patient with LPI. Alveolar macrophages and fibroblastic-like mesenchymal cells were obtained from a whole lung lavage (WLL) performed on the same patient. System y+L activity was determined measuring the 1-min uptake of [3H]-arginine under discriminating conditions. Gene expression was evaluated through qRT-PCR. Results We have found that: 1) system y+L activity is markedly lowered in monocytes and alveolar macrophages from the LPI patient, because of the prevailing expression of SLC7A7/y+LAT1 in these cells; 2) on the contrary, fibroblasts isolated from the same patient do not display the transport defect due to compensation by the SLC7A6/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of SLC7A7, suggesting that the gene is a target of the cytokine; 4) GM-CSF-induced differentiation of LPI monocytes is comparable to that of normal cells, demonstrating that GM-CSF signalling is unaltered; 5) general and respiratory conditions of the patient, along with PAP-associated parameters, markedly improved after GM-CSF therapy through aerosolization. Conclusions Monocytes and macrophages, but not fibroblasts, derived from a LPI patient clearly display the defect in system y+L-mediated arginine transport. The different transport phenotypes are referable to the relative levels of expression of SLC7A7 and SLC7A6. Moreover, the expression of SLC7A7 is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.

At present, the central role played by arginine in the modulation of the inflammatory cellular responses is well-recognized, and many pro-inflammatory stimuli are known to modulate the expression and activity of its transmembrane transporters. In this regard, we have addressed the effects of bacterial flagellin from Pseudomonas aeruginosa (FLA-PA) on the uptake of the amino acid in human epithelial respiratory cells. Among the arginine transporters, only ATB0,+, y+L, and y+ were operative in bronchial epithelial Calu-3 cells under control conditions; however, only the expression and activity of ATB0,+ were stimulated upon incubation with flagellin, whereas those of systems y+L and y+ were not stimulated. As a result, this induction, in turn, led to an increase in the intracellular content of arginine without making any change to its metabolic pathway. In addition, flagellin upregulated the amount of other amino acids substrates of ATB0,+, in particular, all the essential amino acids, such as valine, isoleucine, and leucine, along with the non-essential glutamine. At the molecular level, these effects were directly referable to the stimulation of a toll-like receptor-5 (TLR5) signaling pathway and to the induction of nuclear factor-κB (NF-κB) transcription factor. An induction of ATB0,+ expression has been observed also in EpiAirway™, a model of primary human normal tracheal-bronchial epithelial cells that mimics the in vitro pseudostratified columnar epithelium of the airways. In this tissue model, the incubation with flagellin is associated with the upregulation of messenger RNAs (mRNAs) for the chemokine IL-8 and for the cytokines IL-6 and interleukin-1β (IL-1β); as for the latter, a marked secretion in the extracellular medium was also observed due to the concomitant activation of caspase-1. The overall findings indicate that, in human respiratory epithelium, flagellin promotes cellular responses associating the increase of intracellular amino acids through ATB0,+ with the activation of the inflammasome. Given the role of the ATB0,+ transporter as a delivery system for bronchodilators in human airway epithelial cells, its induction under inflammatory conditions gains particular relevance in the field of respiratory pharmacology. [ABSTRACT FROM AUTHOR]

In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by β-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery. [ABSTRACT FROM AUTHOR]

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein‐rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L‐mediated arginine transport is mainly accounted for by y+LAT1 in monocyte‐derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco‐2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco‐2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease. [ABSTRACT FROM AUTHOR]

At present, the central role played by arginine in the modulation of the inflammatory cellular responses is well-recognized, and many pro-inflammatory stimuli are known to modulate the expression and activity of its transmembrane transporters. In this regard, we have addressed the effects of bacterial flagellin from Pseudomonas aeruginosa (FLA-PA) on the uptake of the amino acid in human epithelial respiratory cells. Among the arginine transporters, only ATB 0,+ , y + L, and y + were operative in bronchial epithelial Calu-3 cells under control conditions; however, only the expression and activity of ATB 0,+ were stimulated upon incubation with flagellin, whereas those of systems y + L and y + were not stimulated. As a result, this induction, in turn, led to an increase in the intracellular content of arginine without making any change to its metabolic pathway. In addition, flagellin upregulated the amount of other amino acids substrates of ATB 0,+ , in particular, all the essential amino acids, such as valine, isoleucine, and leucine, along with the non-essential glutamine. At the molecular level, these effects were directly referable to the stimulation of a toll-like receptor-5 (TLR5) signaling pathway and to the induction of nuclear factor-κB (NF-κB) transcription factor. An induction of ATB 0,+ expression has been observed also in EpiAirway™, a model of primary human normal tracheal-bronchial epithelial cells that mimics the in vitro pseudostratified columnar epithelium of the airways. In this tissue model, the incubation with flagellin is associated with the upregulation of messenger RNAs (mRNAs) for the chemokine IL-8 and for the cytokines IL-6 and interleukin-1β (IL-1β); as for the latter, a marked secretion in the extracellular medium was also observed due to the concomitant activation of caspase-1. The overall findings indicate that, in human respiratory epithelium, flagellin promotes cellular responses associating the increase of intracellular amino acids through ATB 0,+ with the activation of the inflammasome. Given the role of the ATB 0,+ transporter as a delivery system for bronchodilators in human airway epithelial cells, its induction under inflammatory conditions gains particular relevance in the field of respiratory pharmacology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Barilli, Visigalli, Ferrari, Borsani, Dall'Asta and Rotoli.)

Human umbilical vein endothelial cells transport arginine through two Na(+)-independent systems. System y(+)L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na(+), and referable to the expression of SLC7A6/y(+)LAT2, SLC7A7/y(+)LAT1, and SLC3A2/4F2hc. System y(+) is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y(+). Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y(+)L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-alpha, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-alpha or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production.