Skin biomarkers predict the development of food allergy in early life.

BACKGROUND: Food allergy (FA) often occurs in early childhood with and without atopic dermatitis (AD). FA can be severe and even fatal. For primary prevention, it is important to find early biomarkers to predict the future onset of FA before any clinical manifestations. OBJECTIVE: Our aim was to find early predictors of future onset of FA in the stratum corneum (SC). METHODS: Skin tape strips were collected from the forearm of newborns (n = 129) at age 2 months, before any signs of clinical FA or AD. Children were clinically monitored until they reached age 2 years to confirm the presence or absence of FA and AD. Skin tape strips were subjected to lipidomic analyses by liquid chromatography-tandem mass spectrometry and cytokine determination by Meso Scale Discovery U-Plex assay. RESULTS: Overall, 9 of 129 infants (7.0%) developed FA alone and 9 of 129 infants (7.0%) developed FA concomitantly with AD. In the stratum corneum of children with future FA and concomitant AD and FA, absolute amounts of unsaturated (N24:1)(C18-sphingosine)ceramide and (N26:1)(C18-sphingosine)ceramide and their relative percentages within the molecular group were increased compared with the amounts and percentages in healthy children, with P values ranging from less than .01 to less than .05 according to ANOVA. The children with future AD had normal levels of these molecules. IL-33 level was upregulated in those infants with future FA but not in those with future AD, whereas thymic stromal lymphopoietin was upregulated in those with future AD but not in those with future FA. Logistic regression analysis revealed strong FA predicting power for the combination of dysregulated lipids and cytokines, with an odds ratio reaching 101.4 (95% CI = 5.4-1910.6). CONCLUSION: Noninvasive skin tape strip analysis at age 2 months can identify infants at risk of FA in the future.

Analytical challenges in omics research on asthma and allergy: A National Institute of Allergy and Infectious Diseases workshop.

Studies of asthma and allergy are generating increasing volumes of omics data for analysis and interpretation. The National Institute of Allergy and Infectious Diseases (NIAID) assembled a workshop comprising investigators studying asthma and allergic diseases using omics approaches, omics investigators from outside the field, and NIAID medical and scientific officers to discuss the following areas in asthma and allergy research: genomics, epigenomics, transcriptomics, microbiomics, metabolomics, proteomics, lipidomics, integrative omics, systems biology, and causal inference. Current states of the art, present challenges, novel and emerging strategies, and priorities for progress were presented and discussed for each area. This workshop report summarizes the major points and conclusions from this NIAID workshop. As a group, the investigators underscored the imperatives for rigorous analytic frameworks, integration of different omics data types, cross-disciplinary interaction, strategies for overcoming current limitations, and the overarching goal to improve scientific understanding and care of asthma and allergic diseases.

Endotypes of atopic dermatitis and food allergy.

Atopic dermatitis (AD) and food allergy (FA) are strongly associated, with one-third of children with AD developing concomitant FA. Epithelial barrier dysfunction is important in both conditions. Genetic factors, such as filaggrin mutations and IL-4 receptor alpha chain polymorphisms, are linked to increased risk. In addition, several environmental exposures lead to reduced filaggrin and contribute to skin barrier dysfunction. Staphylococcus aureus colonization appears to contribute to AD and FA, as well as activating the type 2 immune response. Comprehensive multiomic studies using skin tape stripping have identified distinct atopic endotypes with unique characteristics of the stratum corneum lipids, proteins, S aureus abundance, and type 2 cytokine expression. Our new understanding of AD and FA presents an area of opportunity to move toward improved diagnosis and prevention of atopy.

Stratum corneum lipid and cytokine biomarkers at age 2 months predict the future onset of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) commonly occurs in children and can progress into severe phenotypes or atopic march, causing significant impairment in quality of life. It is important to find early biomarkers of future onset of AD before any clinical manifestations. OBJECTIVE: We sought to find early predictors of future onset of AD in skin stratum corneum (SC). METHODS: Skin tape strips were collected from the forearm of newborns (n = 111) with and without family history of atopic diseases at the age of 2 months before any signs of clinical AD. Children were clinically monitored until they reached age 2 years to ensure the presence or absence of AD. Skin tape strips were subjected to lipidomic analyses by the liquid chromatography electrospray ionization tandem mass spectrometry and cytokine determination by Meso Scale Discovery U-Plex assay. RESULTS: Overall, 22 of 74 (29.7%) and 5 of 37 (13.5%) infants developed AD in the risk group and the control group, respectively. In the SC of future AD children, protein-bound ceramides were decreased (P < .001), whereas unsaturated sphingomyelin species (P < .0001) and "short-chain" nonhydroxy fatty acid sphingosine and alpha-hydroxy fatty acid sphingosine ceramides were elevated (P < .01 and .05, respectively) as compared with healthy children. Thymic stromal lymphopoietin and IL-13 levels were increased in the SC of future AD subjects (by 74.5% and 78.3%, P = .0022 and P < .0001, respectively). Multivariable logistic regression analysis revealed strong AD predicting power of the combination of family history, type 2 cytokines, and dysregulated lipids, with an odds ratio reaching 54.0 (95% CI, 9.2-317.5). CONCLUSIONS: Noninvasive skin tape strip analysis at age 2 months can identify asymptomatic children at risk of future AD development with a high probability.

Staphylococcus aureus causes aberrant epidermal lipid composition and skin barrier dysfunction.

BACKGROUND: Staphylococcus (S) aureus colonization is known to cause skin barrier disruption in atopic dermatitis (AD) patients. However, it has not been studied how S. aureus induces aberrant epidermal lipid composition and skin barrier dysfunction. METHODS: Skin tape strips (STS) and swabs were obtained from 24 children with AD (6.0 ± 4.4 years) and 16 healthy children (7.0 ± 4.5 years). Lipidomic analysis of STS samples was performed by mass spectrometry. Skin levels of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) were evaluated. The effects of MSSA and MRSA were evaluated in primary human keratinocytes (HEKs) and organotypic skin cultures. RESULTS: AD and organotypic skin colonized with MRSA significantly increased the proportion of lipid species with nonhydroxy fatty acid sphingosine ceramide with palmitic acid ([N-16:0 NS-CER], sphingomyelins [16:0-18:0 SM]), and lysophosphatidylcholines [16:0-18:0 LPC], but significantly reduced the proportion of corresponding very long-chain fatty acids (VLCFAs) species (C22-28) compared to the skin without S. aureus colonization. Significantly increased transepidermal water loss (TEWL) was found in MRSA-colonized AD skin. S. aureus indirectly through interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, and IL-33 inhibited expression of fatty acid elongase enzymes (ELOVL3 and ELOVL4) in HEKs. ELOVL inhibition was more pronounced by MRSA and resulted in TEWL increase in organotypic skin. CONCLUSION: Aberrant skin lipid profiles and barrier dysfunction are associated with S. aureus colonization in AD patients. These effects are attributed to the inhibition of ELOVLs by S. aureus-induced IL-1ß, TNF-α, IL-6, and IL-33 seen in keratinocyte models and are more prominent in MRSA than MSSA.

Signaling sphingolipids are biomarkers for atopic dermatitis prone to disseminated viral infections.

BACKGROUND: Life-threatening viral diseases such as eczema herpeticum (EH) and eczema vaccinatum (EV) occur in <5% of individuals with atopic dermatitis (AD). The diagnosis of AD, however, excludes all individuals with AD from smallpox vaccination. OBJECTIVES: We sought to identify circulatory and skin lipid biomarkers associated with EH and EV. METHODS: Stratum corneum and plasma samples from 15 subjects with AD and a history of EH, 13 age- and gender-matched subjects with AD and without EH history, and 13 healthy nonatopic (NA) controls were analyzed by liquid chromatography tandem mass spectrometry for sphingolipid content. Sphingosine-1-phosphate (S1P) and ceramide levels were validated in plasma samples from the Atopic Dermatitis Vaccinia Network/Atopic Dermatitis Research Network repository (12 NA, 12 AD, 23 EH) and plasma from 7 subjects with EV and 7 matched subjects with AD. S1P lyase was downregulated in human primary keratinocytes to evaluate its effect on herpes simplex virus 1 (HSV-1) replication in vitro. RESULTS: The stratum corneum of patients with EH demonstrated significantly higher levels of free sphingoid bases than those in patients who were NA, indicating enhanced sphingolipid turnover in keratinocytes (P < .05). Plasma from 2 independent cohorts of patients with EH had a significantly increased S1P/ceramide ratio in subjects with EH versus those with AD and or who were NA (P < .01). The S1P level in plasma from subjects with EV was twice the level in plasma from subjects with AD (mean = 1,533 vs 732 pmol/mL; P < .001). Downregulation of S1P lyase expression with silencing RNA led to an increased S1P level and doubled HSV-1 titer in keratinocytes. CONCLUSIONS: Our data point to long-term abnormalities in the S1P signaling system as a biomarker for previous disseminated viral diseases and a potential treatment target in recurring infections.

Group 1 CD1-restricted T cells contribute to control of systemic Staphylococcus aureus infection.

Staphylococcus aureus (SA) is the causative agent of both skin/soft tissue infections as well as invasive bloodstream infections. Though vaccines have been developed to target both humoral and T cell-mediated immune responses against SA, they have largely failed due to lack of protective efficacy. Group 1 CD1-restricted T cells recognize lipid rather than peptide antigens. Previously found to recognize lipids derived from cell wall of Mycobacterium tuberculosis (Mtb), these cells were associated with protection against Mtb infection in humans. Using a transgenic mouse model expressing human group 1 CD1 molecules (hCD1Tg), we demonstrate that group 1 CD1-restricted T cells can recognize SA-derived lipids in both immunization and infection settings. Systemic infection of hCD1Tg mice showed that SA-specific group 1 CD1-restricted T cell response peaked at 10 days post-infection, and hCD1Tg mice displayed significantly decreased kidney pathology at this time point compared with WT control mice. Immunodominant SA lipid antigens recognized by group 1 CD1-restricted T cells were comprised mainly of cardiolipin and phosphatidyl glycerol, with little contribution from lysyl-phosphatidyl glycerol which is a unique bacterial lipid not present in mammals. Group 1 CD1-restricted T cell lines specific for SA lipids also conferred protection against SA infection in the kidney after adoptive transfer. They were further able to effectively control SA replication in vitro through direct antigen presentation by group 1 CD1-expressing BMDCs. Together, our data demonstrate a previously unknown role for group 1 CD1-restricted SA lipid-specific T cells in the control of systemic MRSA infection.

Dupilumab significantly improves skin barrier function in patients with moderate-to-severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is characterized by abnormal skin lipids that are largely driven by hyperactivated type 2 immune responses. The antibody to the α-subunit of interleukin (IL)-4 receptor, dupilumab, was recently approved to treat AD and demonstrated strong efficacy. However, the role of dupilumab therapy in the regulation of skin barrier structure and function has not been fully explored. METHODS: We have evaluated the content of lipids and transepidermal water loss (TEWL) in lesional and non-lesional skin of adults and adolescents with moderate-to-severe AD over the course of 16-week treatment with dupilumab and compared those values with that of matched healthy volunteers. RESULTS: Dupilumab treatment provided a significant decrease in TEWL in AD lesions, lowering it almost to the levels seen in the skin of healthy subjects. Blocking IL-4/IL-13 signaling with dupilumab normalized lipid composition (decreased levels of ceramides with non-hydroxy fatty acids and C18-sphingosine and increased the level of esterified omega-hydroxy fatty acid-containing ceramides) and increased ceramide chain length in lesional as well as non-lesional stratum corneum of AD patients. Partial changes for these parameters were already observed after 2 weeks, with a full response achieved after 8 weeks of dupilumab treatment. CONCLUSIONS: Inhibition of IL-4/IL-13 signaling by dupilumab allows restoration of skin lipid composition and barrier function in patients with moderate-to-severe AD.

Unique skin abnormality in patients with peanut allergy but no atopic dermatitis.

BACKGROUND: The nonlesional skin of children with atopic dermatitis (AD) with peanut allergy (PA) is associated with increased transepidermal water loss; low urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), both of which are filaggrin breakdown products; and a reduced ratio of esterified ω-hydroxy fatty acid sphingosine ceramides (EOS-CERs) to nonhydroxy fatty acid sphingosine ceramides (NS-CERs) in the skin. The skin barrier of subjects with PA without AD (AD-PA+) has not been studied. OBJECTIVE: Our aim was to explore whether AD-PA+ is associated with skin barrier abnormalities. METHODS: A total of 33 participants were enrolled, including 13 AD-PA+, 9 AD+PA+, and 11 nonatopic (NA) participants. RESULTS: The PCA content in the stratum corneum of AD-PA+ subjects was significantly reduced versus that in NA subjects (median level, 67 vs 97 µg/mg protein [P = .028]). The ratio between cis- and trans-UCA decreased significantly from being highest in the NA group (1.62) to lowest in AD+PA+ group (0.07 [P < .001 vs in the NA group; P = .006 vs in the AD-PA+ group]), with the AD-PA+ group having an intermediate cis/trans-UCA ratio (1.17 [P = .024 vs in the NA group]). The TEWL in AD-PA+ subjects did not differ from that in the group with NA skin. Interestingly, AD-PA+ subjects had an increased EOS/NS-CER ratio versus that in the group of subjects with NA skin (1.9 vs 1.3 [P = .008]), whereas the AD+PA+ group had a decreased proportion of EOS-CERs (0.8 [P = .001] vs in the AD-PA+ group). CONCLUSION: Our data demonstrate that irrespective of AD, PA is associated with decreased skin cis-UCA and PCA content. An increase in skin EOS-CER/NS-CER ratio separates the AD-PA+ group from the AD+PA+ and NA groups.

Altered Macrophage Function Associated with Crystalline Lung Inflammation in Acid Sphingomyelinase Deficiency.

Deficiency of ASM (acid sphingomyelinase) causes the lysosomal storage Niemann-Pick disease (NPD). Patients with NPD type B may develop progressive interstitial lung disease with frequent respiratory infections. Although several investigations using the ASM-deficient (ASMKO) mouse NPD model revealed inflammation and foamy macrophages, there is little insight into the pathogenesis of NPD-associated lung disease. Using ASMKO mice, we report that ASM deficiency is associated with a complex inflammatory phenotype characterized by marked accumulation of monocyte-derived CD11b+ macrophages and expansion of airspace/alveolar CD11c+ CD11b- macrophages, both with increased size, granularity, and foaminess. Both the alternative and classical pathways were activated, with decreased in situ phagocytosis of opsonized (Fc-coated) targets, preserved clearance of apoptotic cells (efferocytosis), secretion of Th2 cytokines, increased CD11c+/CD11b+ cells, and more than a twofold increase in lung and plasma proinflammatory cytokines. Macrophages, neutrophils, eosinophils, and noninflammatory lung cells of ASMKO lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, which formed large eosinophilic polygonal Charcot-Leyden-like crystals. In addition to providing insight into novel features of lung inflammation that may be associated with NPD, our report provides a novel connection between ASM and the development of crystal-associated lung inflammation with alterations in macrophage biology.

Ceramide and sphingosine-1 phosphate in COPD lungs.

Studies of chronic obstructive pulmonary disease (COPD) using animal models and patient plasma indicate dysregulation of sphingolipid metabolism, but data in COPD lungs are sparse. Mass spectrometric and immunostaining measurements of lungs from 69 COPD, 16 smokers without COPD and 13 subjects with interstitial lung disease identified decoupling of lung ceramide and sphingosine-1 phosphate (S1P) levels and decreased sphingosine kinase-1 (SphK1) activity in COPD. The correlation of ceramide abundance in distal COPD lungs with apoptosis and the inverse correlation between SphK1 activity and presence of emphysema suggest that disruption of ceramide-to-S1P metabolism is an important determinant of emphysema phenotype in COPD.

Impairment of Flow-Sensitive Inwardly Rectifying K+ Channels via Disruption of Glycocalyx Mediates Obesity-Induced Endothelial Dysfunction.

OBJECTIVE: To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial Kir (inwardly rectifying K+) channels. Approach and Results: Endothelial dysfunction, observed as reduced dilations to flow, occurred after feeding mice a high-fat, Western diet for 8 weeks. The functional downregulation of endothelial Kir2.1 using dominant-negative Kir2.1 construct resulted in substantial reductions in the response to flow in mesenteric arteries of lean mice, whereas no effect was observed in arteries of obese mice. Overexpressing wild-type-Kir2.1 in endothelium of arteries from obese mice resulted in full recovery of the flow response. Exposing freshly isolated endothelial cells to fluid shear during patch-clamp electrophysiology revealed that the flow-sensitivity of Kir was virtually abolished in cells from obese mice. Atomic force microscopy revealed that the endothelial glycocalyx was stiffer and the thickness of the glycocalyx layer reduced in arteries from obese mice. We also identified that the length of the glycocalyx is critical to the flow-activation of Kir. Overexpressing Kir2.1 in endothelium of arteries from obese mice restored flow- and heparanase-sensitivity, indicating an important role for heparan sulfates in the flow-activation of Kir. Furthermore, the Kir2.1-dependent component of flow-induced vasodilation was lost in the endothelium of resistance arteries of obese humans obtained from biopsies collected during bariatric surgery. CONCLUSIONS: We conclude that obesity-induced impairment of flow-induced vasodilation is attributed to the loss of flow-sensitivity of endothelial Kir channels and propose that the latter is mediated by the biophysical alterations of the glycocalyx.

Cutaneous barrier dysfunction in allergic diseases.

The fundamental defect(s) that drives atopic dermatitis (AD) remains controversial. "Outside in" proponents point to the important association of filaggrin gene mutations and other skin barrier defects with AD. The "inside out" proponents derive support from evidence that AD occurs in genetic animal models with overexpression of type 2 immune pathways in their skin, and humans with gain-of-function mutations in their type 2 response develop severe AD. The observation that therapeutic biologics, targeting type 2 immune responses, can reverse AD provides compelling support for the importance of "inside out" mechanisms of AD. In this review, we propose a central role for epithelial cell dysfunction that accounts for the dual role of skin barrier defects and immune pathway activation in AD. The complexity of AD has its roots in the dysfunction of the epithelial barrier that allows the penetration of allergens, irritants, and microbes into a cutaneous milieu that facilitates the induction of type 2 immune responses. The AD phenotypes and endotypes that result in chronic skin inflammation and barrier dysfunction are modified by genes, innate/adaptive immune responses, and different environmental factors that cause skin barrier dysfunction. There is also compelling evidence that skin barrier dysfunction can alter the course of childhood asthma, food allergy, and allergic rhinosinusitis. Effective management of AD requires a multipronged approach, not only restoring cutaneous barrier function, microbial flora, and immune homeostasis but also enhancing skin epithelial differentiation.

Skin tape proteomics identifies pathways associated with transepidermal water loss and allergen polysensitization in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) and food allergy (FA) are associated with skin barrier dysfunction. OBJECTIVE: Skin biomarkers are needed for skin barrier interventions studies. METHODS: In this study, skin tape strip (STS) samples were collected from nonlesional skin of 62 children in AD FA+, AD FA-, and nonatopic groups for mass spectrometry proteomic analysis. transepidermal water loss and allergic sensitization were assessed. STS proteomic analysis results were validated in an independent cohort of 41 adults with AD with and without FA versus nonatopic controls. RESULTS: A group of 45 proteins was identified as a principal component 1 (PC1) with the highest expression in AD FA+ STSs. This novel set of STS proteins was highly correlative to skin transepidermal water loss and allergic sensitization. PC1 proteins included keratin intermediate filaments; proteins associated with inflammatory responses (S100 proteins, alarmins, protease inhibitors); and glycolysis and antioxidant defense enzymes. Analysis of PC1 proteins expression in an independent adult AD cohort validated differential expression of STS PC1 proteins in the skin of adult patients with AD with the history of clinical reactions to peanut. CONCLUSIONS: STS analysis of nonlesional skin of AD children identified a cluster of proteins with the highest expression in AD FA+ children. The differential expression of STS PC1 proteins was confirmed in a replicate cohort of adult AD patients with FA to peanut, suggesting a unique STS proteomic endotype for AD FA+ that persists into adulthood. Collectively, PC1 proteins are associated with abnormalities in skin barrier integrity and may increase the risk of epicutaneous sensitization to food allergens.

Neonatal therapy with PF543, a sphingosine kinase 1 inhibitor, ameliorates hyperoxia-induced airway remodeling in a murine model of bronchopulmonary dysplasia.

Hyperoxia (HO)-induced lung injury contributes to bronchopulmonary dysplasia (BPD) in preterm newborns. Intractable wheezing seen in BPD survivors is associated with airway remodeling (AWRM). Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling promotes HO-mediated neonatal BPD; however, its role in the sequela of AWRM is not known. We noted an increased concentration of S1P in tracheal aspirates of neonatal infants with severe BPD, and earlier, demonstrated that Sphk1-/- mice showed protection against HO-induced BPD. The role of SPHK1/S1P in promoting AWRM following exposure of neonates to HO was investigated in a murine model. Therapy using PF543, the specific SPHK1 inhibitor, during neonatal HO reduced alveolar simplification followed by reduced AWRM in adult mice. This was associated with reduced airway hyperreactivity to intravenous methacholine. Neonatal HO exposure was associated with increased expression of SPHK1 in lung tissue of adult mice, which was reduced with PF543 therapy in the neonatal stage. This was accompanied by amelioration of HO-induced reduction of E-cadherin in airway epithelium. This may be suggestive of arrested partial epithelial mesenchymal transition (EMT) induced by HO. In vitro studies using human primary airway epithelial cells (HAEpCs) showed that SPHK1 inhibition or deletion restored HO-induced reduction in E-cadherin and reduced formation of mitochondrial reactive oxygen species (mtROS). Blocking mtROS with MitoTempo attenuated HO-induced partial EMT of HAEpCs. These results collectively support a therapeutic role for PF543 in preventing HO-induced BPD in neonates and the long-term sequela of AWRM, thus conferring a long-term protection resulting in improved lung development and function.

Elevated truncated oxidized phospholipids as a factor exacerbating ALI in the aging lungs.

As mechanisms controlling redox homeostasis become impaired with aging, exaggerated oxidant stress may cause disproportional oxidation of cell membranes and circulating phospholipids (PLs), leading to the formation of truncated oxidized PL products (Tr-OxPLs), which exhibit deleterious effects. This study investigated the role of elevated Tr-OxPLs as a factor exacerbating inflammation and lung barrier dysfunction in an animal model of aging. Mass spectrometry analysis of Tr-OxPL species in young (2-4 mo) and aging (18-24 mo) mice revealed elevated basal levels of several products [1-palmitoyl-2-(5-oxovaleroyl)- sn-glycero-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl- sn-glycero-phosphocholine, lysophosphocholine, 1-palmitoyl-2-(9-oxo-nonanoyl)- sn-glycero-3-phosphocholine, 1-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine, O-1-O-palmitoyl-2-O-(5,8-dioxo-8-hydroxy-6-octenoyl)-l-glycero-3-phosphocholine, and others] in the aged lungs. An intratracheal (i.t.) injection of bacterial LPS caused increased generation of Tr-OxPLs in the lungs but not in the liver, with higher levels detected in the aged group. In addition, OxPLs clearance from the lung tissue after LPS challenge was delayed in the aged group. The impact of Tr-OxPLs on endothelial cell (EC) barrier compromise under inflammatory conditions was further evaluated in the 2-hit cell culture model of acute lung injury (ALI). EC barrier dysfunction caused by cell treatment with a cytokine mixture (CM) was augmented by cotreatment with low-dose Tr-OxPLs, which did not significantly affect endothelial function when added alone. Deleterious effects of Tr-OxPLs on inflamed ECs stimulated with CM were associated with further weakening of cell junctions and more robust EC hyperpermeability. Aged mice injected intratracheally with TNF-α exhibited a more pronounced elevation of cell counts and protein content in bronchoalveolar lavage (BAL) samples. Interestingly, intravenous administration of low POVPC doses-which did not affect BAL parameters alone in young mice exposed to i.t. TNF-α challenge-augmented lung injury to the levels observed in aged mice stimulated with TNF-α alone. Inhibition of Tr-OxPL generation by ectopic expression of PL-specific platelet-activating factor acetylhydrolase 2 (PAFAH2) markedly reduced EC dysfunction induced by CM, whereas PAFAH2 pharmacologic inhibition augmented deleterious effects of cytokines on EC barrier function. Moreover, exacerbating effects of PAFAH2 inhibition on TNF-α-induced lung injury were observed in vivo. These results demonstrate an age-dependent increase in Tr-OxPL production under basal conditions and augmented Tr-OxPL generation upon inflammatory stimulation, suggesting a major role for elevated Tr-OxPLs in more severe ALI and delayed resolution in aging lungs.-Ke, Y., Karki, P., Kim, J., Son, S., Berdyshev, E., Bochkov, V. N., Birukova, A. A., Birukov, K. G. Elevated truncated oxidized phospholipids as a factor exacerbating ALI in the aging lungs.

Role of Glucosylceramide in Lung Endothelial Cell Fate and Emphysema.

Rationale: The loss of pulmonary endothelial cells in emphysema is associated with increased lung ceramide. Ceramide perturbations may cause adaptive alterations in other bioactive sphingolipids, with pathogenic implications. We previously reported a negative correlation between emphysema and circulating glycosphingolipids (GSLs). Glucosylceramide (GlcCer), the initial GSL synthesized from ceramide by GCS (GlcCer synthase), is required for embryonic survival, but its role in the lung is unknown.Objectives: To determine if cigarette smoke (CS) alters lung GlcCer and to elucidate the role of GCS in lung endothelial cell fate.Methods: GlcCer was measured by tandem mass spectrometry in BAL fluid of CS- or elastase-exposed mice, and GCS was detected by Western blotting in chronic obstructive pulmonary disease lungs and CS extract-exposed primary human lung microvascular endothelial cells (HLMVECs). The role of GlcCer and GCS on mTOR (mammalian target of rapamycin) signaling, autophagy, lysosomal function, and cell death were studied in HLMVECs with or without CS exposure.Measurements and Main Results: Mice exposed to chronic CS or to elastase, and patients with chronic obstructive pulmonary disease, exhibited significantly decreased lung GlcCer and GCS. In mice, lung GlcCer levels were negatively correlated with airspace size. GCS inhibition in HLMVEC increased lysosomal pH, suppressed mTOR signaling, and triggered autophagy with impaired lysosomal degradation and apoptosis, recapitulating CS effects. In turn, increasing GlcCer by GCS overexpression in HLMVEC improved autophagic flux and attenuated CS-induced apoptosis.Conclusions: Decreased GSL production in response to CS may be involved in emphysema pathogenesis, associated with autophagy with impaired lysosomal degradation and lung endothelial cell apoptosis.

Ceramide Signaling and Metabolism in Pathophysiological States of the Lung.

Following the discovery of ceramide as the central signaling and metabolic relay among sphingolipids, studies of its involvement in lung health and pathophysiology have exponentially increased. In this review, we highlight key studies in the context of recent progress in metabolomics and translational research methodologies. Evidence points toward an important role for the ceramide/sphingosine-1-phosphate rheostat in maintaining lung cell survival, vascular barrier function, and proper host response to airway microbial infections. Sphingosine kinase 1 has emerged as an important determinant of sphingosine-1-phosphate lung levels, which, when aberrantly high, contribute to lung fibrosis, maladaptive vascular remodeling, and allergic asthma. New sphingolipid metabolites have been discovered as potential biomarkers of several lung diseases. Although multiple acute and chronic lung pathological conditions involve perturbations in sphingolipid signaling and metabolism, there are specific patterns, unique sphingolipid species, enzymes, metabolites, and receptors, which have emerged that deepen our understanding of lung pathophysiology and inform the development of new therapies for lung diseases.

Pseudomonas aeruginosa stimulates nuclear sphingosine-1-phosphate generation and epigenetic regulation of lung inflammatory injury.

INTRODUCTION: Dysregulated sphingolipid metabolism has been implicated in the pathogenesis of various pulmonary disorders. Nuclear sphingosine-1-phosphate (S1P) has been shown to regulate histone acetylation, and therefore could mediate pro-inflammatory genes expression. METHODS: Profile of sphingolipid species in bronchoalveolar lavage fluids and lung tissue of mice challenged with Pseudomonas aeruginosa (PA) was investigated. The role of nuclear sphingosine kinase (SPHK)2 and S1P in lung inflammatory injury by PA using genetically engineered mice was determined. RESULTS: Genetic deletion of Sphk2, but not Sphk1, in mice conferred protection from PA-mediated lung inflammation. PA infection stimulated phosphorylation of SPHK2 and its localisation in epithelial cell nucleus, which was mediated by protein kinase C (PKC) δ. Inhibition of PKC δ or SPHK2 activity reduced PA-mediated acetylation of histone H3 and H4, which was necessary for the secretion of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-α. The clinical significance of the findings is supported by enhanced nuclear localisation of p-SPHK2 in the epithelium of lung specimens from patients with cystic fibrosis (CF). CONCLUSIONS: Our studies define a critical role for nuclear SPHK2/S1P signalling in epigenetic regulation of bacterial-mediated inflammatory lung injury. Targeting SPHK2 may represent a potential strategy to reduce lung inflammatory pulmonary disorders such as pneumonia and CF.

Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

The mechanisms by which lung structural cells survive toxic exposures to cigarette smoke (CS) are not well defined but may involve proper disposal of damaged mitochondria by macro-autophagy (mitophagy), processes that may be influenced by pro-apoptotic ceramide (Cer) or its precursor dihydroceramide (DHC). Human lung epithelial and endothelial cells exposed to CS exhibited mitochondrial damage, signaled by phosphatase and tensin homolog-induced putative kinase 1 (PINK1) phosphorylation, autophagy, and necroptosis. Although cells responded to CS by rapid inhibition of DHC desaturase, which elevated DHC levels, palmitoyl (C16)-Cer also increased in CS-exposed cells. Whereas DHC augmentation triggered autophagy without cell death, the exogenous administration of C16-Cer was sufficient to trigger necroptosis. Inhibition of Cer-generating acid sphingomyelinase reduced both CS-induced PINK1 phosphorylation and necroptosis. When exposed to CS, Pink1-deficient ( Pink1-/-) mice, which are protected from airspace enlargement compared with wild-type littermates, had blunted C16-Cer elevations and less lung necroptosis. CS-exposed Pink1-/- mice also exhibited significantly increased levels of lignoceroyl (C24)-DHC, along with increased expression of Cer synthase 2 ( CerS2), the enzyme responsible for its production. This suggested that a combination of high C24-DHC and low C16-Cer levels might protect against CS-induced necroptosis. Indeed, CerS2-/- mice, which lack C24-DHC at the expense of increased C16-Cer, were more susceptible to CS, developing airspace enlargement following only 1 month of exposure. These results implicate DHCs, in particular, C24-DHC, as protective against CS toxicity by enhancing autophagy, whereas C16-Cer accumulation contributes to mitochondrial damage and PINK1-mediated necroptosis, which may be amplified by the inhibition of C24-DHC-producing CerS2.-Mizumura, K., Justice, M. J., Schweitzer, K. S., Krishnan, S., Bronova, I., Berdyshev, E. V., Hubbard, W. C., Pewzner-Jung, Y., Futerman, A. H., Choi, A. M. K., Petrache, I. Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.