Inhibition of acid sphingomyelinase disrupts LYNUS signaling and triggers autophagy

Activation of the lysosomal ceramide-producing enzyme, acid sphingomyelinase (ASM), by various stresses is centrally involved in cell death and has been implicated in autophagy. We set out to investigate the role of the baseline ASM activity in maintaining physiological functions of lysosomes, focusing on the lysosomal nutrient-sensing complex (LYNUS), a lysosomal membrane-anchored multiprotein complex that includes mammalian target of rapamycin (mTOR) and transcription factor EB (TFEB). ASM inhibition with imipramine or sphingomyelin phosphodiesterase 1 (SMPD1) siRNA in human lung cells, or by transgenic Smpd1+/- haploinsufficiency of mouse lungs, markedly reduced mTOR- and P70-S6 kinase (Thr 389)-phosphorylation and modified TFEB in a pattern consistent with its activation. Inhibition of baseline ASM activity significantly increased autophagy with preserved degradative potential. Pulse labeling of sphingolipid metabolites revealed that ASM inhibition markedly decreased sphingosine (Sph) and Sph-1-phosphate (S1P) levels at the level of ceramide hydrolysis. These findings suggest that ASM functions to maintain physiological mTOR signaling and inhibit autophagy and implicate Sph and/or S1P in the control of lysosomal function

Docosatetraenoyl LPA is elevated in exhaled breath condensate in idiopathic pulmonary fibrosis

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective medical therapies. Recent research has focused on identifying the biological processes essential to the development and progression of fibrosis, and on the mediators driving these processes. Lysophosphatidic acid (LPA), a biologically active lysophospholipid, is one such mediator. LPA has been found to be elevated in bronchoalveolar lavage (BAL) fluid of IPF patients, and through interaction with its cell surface receptors, it has been shown to drive multiple biological processes implicated in the development of IPF. Accordingly, the first clinical trial of an LPA receptor antagonist in IPF has recently been initiated. In addition to being a therapeutic target, LPA also has potential to be a biomarker for IPF. There is increasing interest in exhaled breath condensate (EBC) analysis as a non-invasive method for biomarker detection in lung diseases, but to what extent LPA is present in EBC is not known. Methods: In this study, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess for the presence of LPA in the EBC and plasma from 11 IPF subjects and 11 controls. Results: A total of 9 different LPA species were detectable in EBC. Of these, docosatetraenoyl (22:4) LPA was significantly elevated in the EBC of IPF subjects when compared to controls (9.18 pM vs. 0.34 pM; p = 0.001). A total of 13 different LPA species were detectable in the plasma, but in contrast to the EBC, there were no statistically significant differences in plasma LPA species between IPF subjects and controls. Conclusions: These results demonstrate that multiple LPA species are detectable in EBC, and that 22:4 LPA levels are elevated in the EBC of IPF patients. Further research is needed to determine the significance of this elevation of 22:4 LPA in IPF EBC, as well as its potential to serve as a biomarker for disease severity and/or progression

The Calcineurin-NFATc Pathway Modulates the Lipid Mediators in BAL Fluid Extracellular Vesicles, Thereby Regulating Microvascular Endothelial Cell Barrier Function

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function

Plasma levels of sphingosine 1-phosphate are strongly correlated with haematocrit, but variably restored by red blood cell transfusions

Anaemia and RBC (red blood cell) transfusion may be associated with worse clinical outcomes, especially with longer blood storage duration prior to transfusion. The mechanisms underlying these harmful effects are unknown. RBCs have been proposed to buffer plasma S1P (sphingosine 1-phosphate), a lysophospholipid essential for the maintenance of endothelial integrity and important in the regulation of haematopoietic cell trafficking. The present study examined the effect of anaemia, RBC transfusion and RBC storage duration on plasma S1P levels. Plasma S1P from 30 individuals demonstrated a linear correlation with Hct (haematocrit; R2=0.51, P<0.001) with no evidence for a plateau at Hct values as low as 19%. RBC transfusion in 23 anaemic patients with baseline mean Hct of 22.2±0.34% (value is the mean±S.D.) increased Hct to 28.3±0.6% at 72 h. Despite an Hct increase, RBC transfusion failed to elevate plasma S1P consistently. A trend towards an inverse correlation was observed between RBC storage duration and the post-transfusion increase in plasma S1P. After 30 days of storage, RBC S1P decreased to 19% of that observed in fresh (3–7-day-old) RBC segments. RBC membranes contain low levels of both S1P phosphatase and S1P lyase activities that may account for the decline in S1P levels with storage. Our results support a role for RBCs in buffering plasma S1P and identify a disturbance in the capacity after transfusion. Changes in S1P content may contribute to an RBC storage lesion. Further studies should investigate the clinical significance of alterations in circulating S1P levels and the potential value of enriching stored RBCs with S1P

Stimulation of sphingosine 1-phosphate signaling as an alveolar cell survival strategy in emphysema

RATIONALE: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. OBJECTIVES: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. METHODS: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. MEASUREMENTS AND MAIN RESULTS: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. CONCLUSIONS: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development

Sphingosine 1 Phosphate (S1P) Receptor 1 Is Decreased in Human Lung Microvascular Endothelial Cells of Smokers and Mediates S1P Effect on Autophagy

Destruction of alveoli by apoptosis induced by cigarette smoke (CS) is a major driver of emphysema pathogenesis. However, when compared to cells isolated from non-smokers, primary human lung microvascular endothelial cells (HLMVECs) isolated from chronic smokers are more resilient when exposed to apoptosis-inducing ceramide. Whether this adaptation restores homeostasis is unknown. To better understand the phenotype of HLMVEC in smokers, we interrogated a major pro-survival pathway supported by sphingosine-1-phosphate (S1P) signaling via S1P receptor 1 (S1P1). Primary HLMVECs from lungs of non-smoker or smoker donors were isolated and studied in culture for up to five passages. S1P1 mRNA and protein abundance were significantly decreased in HLMVECs from smokers compared to non-smokers. S1P1 was also decreased in situ in lungs of mice chronically exposed to CS. Levels of S1P1 expression tended to correlate with those of autophagy markers, and increasing S1P (via S1P lyase knockdown with siRNA) stimulated baseline macroautophagy with lysosomal degradation. In turn, loss of S1P1 (siRNA) inhibited these effects of S1P on HLMVECs autophagy. These findings suggest that the anti-apoptotic phenotype of HLMVECs from smokers may be maladaptive, since it is associated with decreased S1P1 expression that may impair their autophagic response to S1P

Structural and functional characterization of endothelial microparticles released by cigarette smoke

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers

Immunologic Profiling of Immune-Related Cutaneous Adverse Events with Checkpoint Inhibitors Reveals Polarized Actionable Pathways

Purpose: Immune-related cutaneous adverse events (ircAEs) occur in ≥50% of patients treated with checkpoint inhibitors (CPI), but mechanisms are poorly understood. Experimental Design: Phenotyping/biomarker analyses were conducted in 200 patients on CPIs (139 with ircAEs, 61 without, control) to characterize their clinical presentation and immunologic endotypes. Cytokines were evaluated in skin biopsies, skin tape strip (STS) extracts and plasma using real-time PCR and Meso Scale Discovery multiplex cytokine assays. Results: Eight ircAE phenotypes were identified: pruritus (26%), maculopapular rash (MPR; 21%), eczema (19%), lichenoid (11%), urticaria (8%), psoriasiform (6%), vitiligo (5%), and bullous dermatitis (4%). All phenotypes showed skin lymphocyte and eosinophil infiltrates. Skin biopsy PCR revealed the highest increase in IFN-gamma mRNA in patients with lichenoid (p&lt;0.0001) and psoriasiform dermatitis (p&lt;0.01) as compared to patients without ircAEs, while the highest IL-13 mRNA levels were detected in the eczema (p&lt;0.0001, compared to control). IL-17A mRNA was selectively increased in psoriasiform (p&lt;0.001), lichenoid (p&lt;0.0001), bullous dermatitis (p&lt;0.05) and MPR (p&lt;0.001), compared to control. Distinct cytokine profiles were confirmed in STS and plasma. Analysis determined increased skin/plasma IL-4 cytokine in pruritus, skin IL-13 in eczema, plasma IL-5 and IL-31 in eczema and urticaria, and mixed-cytokine pathways in MPR. Broad inhibition via corticosteroids or type 2-cytokine targeted inhibition resulted in clinical benefit in these ircAEs. In contrast, significant skin upregulation of type 1/type 17 pathways was found in psoriasiform, lichenoid, bullous dermatitis, and type 1 activation in vitiligo. Conclusions: Distinct immunologic ircAE endotypes suggest actionable targets for precision medicine-based interventions

Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility