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.

Background. Emerging evidences suggest that in severe COVID-19, multi-organ failure is associated with a hyperinflammatory state (the so-called “cytokine storm”) in combination with the development of a prothrombotic state. The central role of endothelial dysfunction in the pathogenesis of the disease is to date accepted, but the precise mechanisms underlying the associated coagulopathy remain unclear. Whether the alterations in vascular homeostasis directly depend upon the SARS-CoV-2 infection of endothelial cells or, rather, occur secondarily to the activation of the inflammatory response is still a matter of debate. Here, we address the effect of the SARS-CoV-2 spike S1 protein on the activation of human lung microvascular endothelial cells (HLMVEC). In particular, the existence of an endothelium-macrophage crosstalk in the response to the spike protein has been explored. Methods and Results. The effect of the spike protein is addressed in human lung microvascular endothelial cells (HLMVEC), either directly or after incubation with a conditioned medium (CM) of human monocyte-derived macrophages (MDM) previously activated by the spike S1 protein (CM-MDM). Both MDM and HLMVEC are activated in response to the S1 protein, with an increased expression of pro-inflammatory mediators. However, when HLMVEC are exposed to CM-MDM, an enhanced cell activation occurs in terms of the expression of adhesion molecules, pro-coagulant markers, and chemokines. Under this experimental condition, ICAM-1 and VCAM-1, the chemokines CXCL8/IL-8, CCL2/MCP1, and CXCL10/IP-10 as well as the protein tissue factor (TF) are markedly induced. Instead, a decrease of thrombomodulin (THBD) is observed. Conclusion. Our data suggest that pro-inflammatory mediators released by spike-activated macrophages amplify the activation of endothelial cells, likely contributing to the impairment of vascular integrity and to the development of a pro-coagulative endothelium.

The ATP-binding cassette (ABC) transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) play a crucial role in the translocation of a broad range of drugs; data about their expression and activity in lung tissue are controversial. Here, we address their expression, localization and function in EpiAirway™, a three-dimensional (3D)-model of human airways; Calu-3 cells, a representative in vitro model of bronchial epithelium, are used for comparison. Transporter expression has been evaluated with RT-qPCR and Western blot, the localization with immunocytochemistry, and the activity by measuring the apical-to-basolateral and basolateral-to-apical fluxes of specific substrates in the presence of inhibitors. EpiAirway™ and Calu-3 cells express high levels of MRP1 on the basolateral membrane, while they profoundly differ in terms of BCRP and MDR1: BCRP is detected in EpiAirway™, but not in Calu-3 cells, while MDR1 is expressed and functional only in fully-differentiated Calu-3; in EpiAirway™, MDR1 expression and activity are undetectable, consistently with the absence of the protein in specimens from human healthy bronchi. In summary, EpiAirway™ appears to be a promising tool to study the mechanisms of drug delivery in the bronchial epithelium and to clarify the role of ABC transporters in the modulation of the bioavailability of administered drugs.

Background. Clinical and experimental evidence point to a dysregulated immune response caused by SARS-CoV-2 as the primary mechanism of lung disease in COVID-19. However, the pathogenic mechanisms underlying COVID-19-associated ARDS (Acute Respiratory Distress Syndrome) remain incompletely understood. This study aims to explore the inflammatory responses of alveolar epithelial cells to either the spike S1 protein or to a mixture of cytokines secreted by S1-activated macrophages. Methods and Results. The exposure of alveolar A549 cells to supernatants from spike-activated macrophages caused a further release of inflammatory mediators, with IL-8 reaching massive concentrations. The investigation of the molecular pathways indicated that NF-kB is involved in the transcription of IP-10 and RANTES, while STATs drive the expression of all the cytokines/chemokines tested, with the exception of IL-8 which is regulated by AP-1. Cytokines/chemokines produced by spike-activated macrophages are also likely responsible for the observed dysfunction of barrier integrity in Human Alveolar Epithelial Lentivirus-immortalized cells (hAELVi), as demonstrated by an increased permeability of the monolayers to mannitol, a marked decrease of TEER and a disorganization of claudin-7 distribution. Conclusion. Upon exposure to supernatants from S1-activated macrophages, A549 cells act both as targets and sources of cytokines/chemokines, suggesting that alveolar epithelium along with activated macrophages may orchestrate lung inflammation and contribute to alveolar injury, a hallmark of ARDS.

The purpose of this study was to examine the effect of the JAK-STAT inhibitor baricitinib on the inflammatory response of human monocyte-derived macrophages (MDM) and endothelial cells upon exposure to the spike S1 protein from SARS-CoV-2. The effect of the drug has been evaluated on the release of cytokines and chemokines from spike-treated MDM, as well as on the activation of endothelial cells (HUVECs) after exposure to conditioned medium collected from spike-activated MDM. Results obtained indicate that, in MDM, baricitinib prevents the S1-dependent phosphorylation of STAT1 and STAT3, along with the induction of IP-10- and MCP-1 secretion; the release of IL-6 and TNFα is also reduced, while all other mediators tested (IL-1β, IL-8, RANTES, MIP-1α and MIP-1β) are not modified. Baricitinib is, instead, poorly effective on endothelial activation when HUVECs are exposed to supernatants from S1-activated macrophages; the induction of VCAM-1, indeed, is not affected by the drug, while that of ICAM-1 is only poorly inhibited. The drug, however, also exerts protective effects on the endothelium by limiting the expression of pro-inflammatory mediators, specifically IL-6, RANTES and IP-10. No effect of baricitinib has been observed on IL-8 synthesis and, consistently, on neutrophils chemiotaxis. Our in vitro findings reveal that the efficacy of baricitinib is limited, with effects mainly focused on the inhibition of the IL-6-mediated inflammatory loop.

In COVID-19, cytokine release syndrome can cause severe lung tissue damage leading to acute respiratory distress syndrome (ARDS). Here, we address the effects of IFNγ, TNFα, IL-1β and IL-6 on the growth arrest of alveolar A549 cells, focusing on the role of the IFN regulatory factor 1 (IRF1) transcription factor. The efficacy of JAK1/2 inhibitor baricitinib has also been tested. A549 WT and IRF1 KO cells were exposed to cytokines for up to 72 h. Cell proliferation and death were evaluated with the resazurin assay, analysis of cell cycle and cycle-regulator proteins, LDH release and Annexin-V positivity; the induction of senescence and senescence-associated secretory phenotype (SASP) was evaluated through β-galactosidase staining and the quantitation of secreted inflammatory mediators. While IL-1 and IL-6 proved ineffective, IFNγ plus TNFα caused a proliferative arrest in A549 WT cells with alterations in cell morphology, along with the acquisition of a secretory phenotype. These effects were STAT and IRF1-dependent since they were prevented by baricitinib and much less evident in IRF1 KO than in WT cells. In alveolar cells, STATs/IRF1 axis is required for cytokine-induced proliferative arrest and the induction of a secretory phenotype. Hence, baricitininb is a promising therapeutic strategy for the attenuation of senescence-associated inflammation.