Neutrophil elastase increases airway ceramide levels via upregulation of serine palmitoyltransferase

Abstract

Altered sphingolipid metabolism is associated with increased inflammation; however, the impact of inflammatory mediators, including neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. Using a well-characterized mouse model of NE oropharyngeal aspiration, we investigated a potential link between NE-induced airway inflammation and increased synthesis of various classes of sphingolipids, including ceramide species. Sphingolipids in bronchoalveolar lavage fluids (BAL) were identified and quantified using reverse-phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis. BAL total and differential cell counts, CXCL1/keratinocyte chemoattractant (KC) protein levels, and high-mobility group box 1 (HMGB1) protein levels were determined. NE exposure increased BAL long-chain ceramides, total cell and neutrophil counts, and upregulated KC and HMGB1. The mRNA and protein levels of serine palmitoyltransferase (SPT) long-chain subunits 1 and 2, the multimeric enzyme responsible for the first, rate-limiting step of de novo ceramide generation, were determined by qRT-PCR and Western analyses, respectively. NE increased lung SPT long-chain subunit 2 (SPTLC2) protein levels but not SPTLC1 and had no effect on mRNA for either subunit. To assess whether de novo ceramide synthesis was required for NE-induced inflammation, myriocin, a SPT inhibitor, or a vehicle control was administered intraperitoneally 2 h before NE administration. Myriocin decreased BAL d_18:1/22:0 and d_18:1/24:1 ceramide, KC, and HMGB1 induced by NE exposure. These results support a feed-forward cycle of NE-generated ceramide and ceramide-driven cytokine signaling that may be a potential target for intervention in lung disease typified by chronic neutrophilic inflammation.

INTRODUCTION

Neutrophil elastase (NE) is an inflammatory mediator that has been identified as a key biomarker of lung disease in cystic fibrosis (CF) (35, 39) and non-CF bronchiectasis (8). NE-triggered degradation of extracellular matrix is associated with the development of emphysema in chronic obstructive pulmonary disease (COPD) (3). Release of proteolytically active NE injures the airway epithelium and triggers the release of other proinflammatory signaling molecules. In vitro studies have shown that NE upregulates IL-8 in human airway epithelial cell culture (26). In mice, NE initiates an inflammatory response, increasing the concentration of proinflammatory cytokines, including keratinocyte chemokine (KC; murine homolog of CXCL8/IL-8), in murine bronchoalveolar lavage fluids (BAL) (44). Concurrently, NE stimulates the release of high-mobility group box 1 (HMGB1) into murine airways (16). HMGB1 is an alarmin that stimulates neutrophilic inflammation and is a biomarker that correlates with CF lung disease severity (9, 23). HMGB1 activation of the receptor for advanced glycation endproducts is a feature of airway inflammation observed in COPD (11).

The hyperinflammatory response to airway infection observed in CF lung disease is associated with altered homeostasis of lipid molecules, including sphingolipids. In CF, this sphingolipid imbalance has been attributed to loss of the cystic fibrosis transmembrane conductance regulator (CFTR) (2). In smokers with COPD, increased levels of several ceramide moieties are associated with worse lung function and increased inflammation (41). Sphingolipids are bioactive lipids, important contributors to the inflammatory response. Sphingolipids such as ceramide, ceramide 1-phosphate (C1P), and sphingosine 1-phosphate (S1P) can regulate inflammation when released by cells (24, 38). Ceramide, in particular, has been observed to be proinflammatory, and recent findings indicate that ceramide can initiate a signaling cascade via the inflammasome, eventually resulting in the activation of caspase-1 and the release of proinflammatory cytokines, including IL-1β and KC (40).

Ceramide is increased in sputum of patients with CF (33) and COPD (41). In the airway epithelium patients with CF, ceramide correlates with NE-positive and myeloperoxidase- positive cells (5). The elevated ceramide levels detected in the lungs from patients with COPD (29, 32) support the association of altered sphingolipid profiles with inflammation and suggest that inflammation may regulate ceramide levels in the airways.

Ceramide can be generated via three potential pathways: de novo synthesis, sphingomyelin hydrolysis, or conversion from complex sphingolipids (Fig. 1) (24). Given the complex web of interrelated pathways that govern sphingolipid production, ceramide synthesis can be carried out by a variety of enzymes (13, 24). The first and rate-limiting step in the de novo biosynthetic pathway is catalyzed by serine palmitoyltransferase (SPT), a multimeric protein composed of several SPT long-chain subunits (SPTLC) and predicted to be an octamer composed of combinations of SPTLC1, -2, and -3 (21). The regulatory mechanisms for SPT currently remain elusive, with some speculation that the regulation of catalytic activity is linked to the ratio of SPTLC subunits within the protein complex (20). ORM (yeast)-like protein isoform 3 (ORMDL3) (6, 28), reticulon-4 (RTN4, NOGO, NOGOB) (7, 36), and several microRNAs (12) have also been reported to modulate SPT activity. Other enzymatic pathways that promote accumulation of ceramide in the airway include neutral sphingomyelinase (SMase), activated by cigarette smoke and associated with increased ceramide levels in COPD (10), activation of acid SMase in rodent models of emphysema (31), and activation of acid SMase with inhibition of acid ceramidase in CF mice (40).

Fig. 1.

Brief summary of ceramide production. Ceramide is produced through three major pathways. The first involves de novo synthesis from serine and palmitoyl-CoA, where serine palmitoyltransferase (SPT) catalyzes the first and rate-limiting step. In the sphingomyelinase pathway, membrane sphingomyelin is hydrolyzed to ceramide through the action of a sphingomyelinase. Last is the salvage pathway, wherein various complex sphingolipids can be broken down to sphingosine before being converted to ceramide by ceramide synthases.

In the current paradigm, ceramide accumulation in CF is attributed to dysregulation of sphingolipid homeostasis due to the loss of functional CFTR (40), which affects both de novo ceramide synthesis and generation of ceramide from sphingomyelin via the SMase pathway. However, on the basis of the correlation of ceramide levels with neutrophilic inflammation (5, 41) and increased ceramide levels in chronic inflammatory airway diseases, as well as a report that porcine pancreatic elastase induced de novo synthesis of ceramide in a mouse model of emphysema (42), we hypothesized that NE could potentially regulate ceramide homeostasis in the lung.

MATERIALS AND METHODS

Mouse model.

Male Balb/C mice 6–8 wk of age (Jackson, Bar Harbor, ME) were maintained and treated as per an approved IACUC protocol at Virginia Commonwealth University (AD10000870). On days 1, 4, and 7, following isoflurane anesthesia, mice were administered NE (50 μg, 42 μM, SE563; Elastin Products, Owensville, MO) or vehicle control by oropharyngeal aspiration. In separate experiments, mice were administered myriocin [SPT inhibitor, 0.3 mg/kg; 35891-70-4; Cayman Chemicals, Ann Arbor, MI (28, 42)] or a vehicle control by intraperitoneal (ip) injection 2 h before aspiration of NE or vehicle control on days 1, 4, and 7. Mice were euthanized with Euthasol (200-071;Virbac, Carros, France) at 4, 8, or 24 h (day 8) following the final aspiration dose on day 7, underwent bronchoalveolar lavage (BAL), and their lungs harvested.

BAL.

After euthanasia, BAL was collected using 1 ml of sterile normal saline. After centrifugation (500 g, 10 min), the supernatant, to be used for cytokine and sphingolipid analyses, was aliquoted and stored at −80°C. Total cell counts and differentials were obtained from the cell pellet (44).

Cytokine analysis.

The concentration of KC in BAL fluid was measured by ELISA (MKC00B; R&D Systems, Minneapolis, MN), per the manufacturer’s instructions.

Lung homogenization.

Lungs were harvested from mice and snap-frozen in liquid nitrogen. Frozen lungs were homogenized in buffer consisting of 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1× protease inhibitor, and 1× phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO) for protein analysis. After homogenization, samples were sonicated three times for 15 s and incubated with agitation for 1 h at 4°C. Homogenates were clarified by centrifugation (16,000 g, 4°C, 30 min). The resulting supernatant was the lung lysate protein and was quantified using a DC Protein Assay (5000116; Bio-Rad, Hercules, CA), as per the manufacturer's instructions.

qRT-PCR.

Total RNA was isolated from tissue homogenate by using TRIzol (15596026; ThermoFisher Scientific, Waltham, MA) and then processed per the manufacturer’s instructions. qRT-PCR was performed to determine whether NE increases expression of SPTLC1 and SPTLC2 [(Mm00447343_m1 and Mm00448871_m1, respectively; ThermoFisher Scientific] mRNA.

Western blot.

Mouse whole lung lysate (50 μg) or mouse BAL (40 μl) was separated by electrophoresis on a 4–20% SDS-PAGE gel (17000436; Bio-Rad Hercules, CA), transferred to a nitrocellulose membrane, and blocked with 5% nonfat milk in 15 mM Tris, 150 mM NaCl, and 0.1% Tween-20 (TBST). Membranes were incubated in mouse monoclonal anti-HMGB1 (sc-56698; 1:5,000 in 5% milk/TBST; Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-SPTLC2 (ab23696; 0.5 µg/ml in 5% milk/TBST; AbCam, Cambridge, UK), rabbit polyclonal anti-SPTLC1 (15376-1-AP; 1:5,000 in 5% milk/TBST; Proteintech, Chicago, IL), mouse monoclonal anti-NOGO (sc-271878; 1:1,000 in 5%, milk/TBST; Santa Cruz Biotechnology) or anti-ORMDL3 (ABN417; 1:1,000 in 2% BSA/TBST; Millipore, Billerica, MA) primary antibody overnight at 4°C, followed by either HRP-conjugated goat anti-mouse antibody (SC-2005, Santa Cruz Biotechnology) or HRP-conjugated goat anti-rabbit antibody (7074,Cell Signaling Technology, Danvers, MA). Antigen-antibody complexes were visualized by chemiluminescence (ECL Plus; RPN2132; GE Healthcare Life Sciences, Piscataway, NJ), per the manufacturer’s instructions. Membranes with whole lung lysate were reprobed with a monoclonal antibody against β-actin (A5441; 1:5,000 in 5% milk/TBST; Sigma, St. Louis, MO) to confirm equivalent protein loading. Densitometry of target bands was determined by ImageJ software (37), normalized to actin, and then compared with average control values in each experiment.

Sphingolipid assessment.

BAL fluid supernatant was assessed for sphingolipid content by the Lipidomics/Metabolomics Core at Virginia Commonwealth University, using reverse-phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) (46).

Statistical analysis.

Quantitation of sphingolipids ceramide, monohexosylceramides, sphingosine, dihydrosphingosine, sphingosine 1-phosphate, dihydrosphingosine 1-phosphate, and sphingomyelin in BAL, BAL total cell numbers differentials, BAL cytokine levels, as well as relative mRNA and protein expression were compared among the treatment groups by use of either Mann-Whitney U-tests (Wilcoxon rank sum test) or, if there were more than two conditions, one-way nonparametric ANOVA analyses (Kruskal-Wallis) with post hoc multiple comparison adjustments using Mann-Whitney U-tests (Wilcoxon rank sum test) (GraphPad Prism, La Jolla, CA). Data are presented as means ± SE, with differences of P < 0.05 considered statistically significant.

RESULTS

Oropharyngeal aspiration of NE increased the levels of ceramide in murine airways.

We used a well-characterized mouse model of intratracheal NE administration that causes increased airway inflammation (16, 44) to test whether NE-induced inflammation increased airway ceramide levels. Mice were administered three doses of NE (50 µg, 42 µl) or vehicle control (42 µl, 0.9% normal saline) on days 1, 4, and 7. At 4 h (n = 4 animals per group), 8 h (n = 4 animals per group), or 24 h (n = 14 control, n = 16 NE) after the final aspiration, mice were euthanized, and lungs and BAL (1 ml) were collected for analysis. Sphingolipid concentrations in BAL were assessed.

We found that sphingolipid concentrations did not significantly vary between control mice and those that were administered NE at 4 or 8 h (data not shown). At 24 h, the concentrations of total ceramide increased in mice that were administered NE compared with vehicle control (P < 0.01; Table 1 and Fig. 2). However, sphingomyelin, monohexosylceramide, and sphingosine concentrations did not differ between these two groups (Fig. 2). An analysis of the different ceramide moieties present revealed that total ceramide increased due to statistically significant increases (P < 0.01) in long-chain ceramides d_18:1/22:0, d_18:1/24:0, and d_18:1/24:1 (Table 1 and Fig. 3).

Table 1.

Levels of ceramide moieties in BAL

Ceramide, pmol/mla	Control	NE
Total	349.34 ± 32.57	738.52 ± 96.90*
d18:1/14:0	5.83 ± 1.35	3.43 ± 0.88
d18:1/16:0	89.31 ± 16.33	110.17 ± 15.30
d18:1/18:1	17.01 ± 2.79	21.33 ± 4.87
d18:1/18:0	8.96 ± 1.36	10.18 ± 1.96
d18:1/20:0	8.41 ± 1.03	13.34 ± 2.23
d18:1/22:0	29.23 ± 4.85	63.81 ± 10.89*
d18:1/24:1	76.91 ± 9.26	227.03 ± 38.19*
d18:1/24:0	98.85 ± 10.66	268.46 ± 41.43*
d18:1/26:1	8.70 ± 1.04	8.79 ± 1.73
d18:1/26:0	3.79 ± 0.72	7.05 ± 0.62*

Values are means ± SE in pmol/ml bronchoalveolar lavage (BAL) fluid.

^aBAL from n = 16 (control), n = 14 neutrophil elastase (NE) mice at 24 h were collected following the last dose of vehicle control or NE, and sphingolipids were identified and quantified using HPLC-ESI/MS/MS.

^*P < 0.05 vs. control animals at the same time point.

Fig. 2.

Total sphingolipid analysis of bronchoalveolar lavage fluids (BAL) from mice treated with control vehicle or NE. Mice were treated with NE (50 µg, 42 µl) or control vehicle by oropharyngeal aspiration on days 1, 4, and 7. BAL (1 ml) was harvested on day 8. Levels of total sphingomyelin (A), ceramide (B), sphingosine (C), and monohexosylceramide (D) in 200 µl of murine BAL, measured by HPLC-ESI-MS/MS, were increased at 24 h (day 8) after final NE administration compared with vehicle control. The data show sphingolipid content of BAL in pmol/ml, summarizing 2 experiments, n = 16 control, n = 14 NE. Data (means ± SE) were compared by Mann-Whitney U- (Wilcoxon rank sum) test; ***P = 0.0008.

Fig. 3.

Analysis of ceramide chain length in BAL of mice treated with control vehicle or NE. Mice were treated with NE or control vehicle on days 1, 4, and 7. BAL was harvested on day 8. Ceramide levels in murine BAL (200 µl) were measured by HPLC-ESI-MS/MS at 24 h after final NE administration compared with vehicle control. Ceramide d_18:1/22:0 (A), d_18:1/24:1 (B), and d_18:1/24:0 (C) moieties were increased. The data show sphingolipid content of BAL in pmol/ml, summarizing 2 experiments, n = 16 control, n = 14 NE. Data are summarized as means ± SE and compared by Mann-Whitney U- (Wilcoxon rank sum) test; **P < 0.01, ***P < 0.001.

NE increased SPTLC2 protein in murine lungs but did not increase SPTLC1 protein or induce transcription of SPTLC1 or SPTLC2 mRNA.

To investigate whether NE stimulated de novo ceramide synthesis by modulating SPT expression, we analyzed the levels of SPTLC1 and SPTLC2 protein present in the lungs of mice treated with NE or vehicle control. SPTLC2 protein was unchanged at the 4- and 8-h time points (data not shown). In contrast, at 24 h after the final aspiration of NE, the level of SPTLC2 protein in whole lung lysates was increased ~2.5-fold (P < 0.001; Fig. 4, C and D). The levels of SPTLC1 protein were decreased compared with control treated mice (Fig. 4, A and B). Notably, the changes in expression of SPTLC2 over time appeared to mirror the changes in ceramide levels (d_18:1/22:0, d_18:1/24:0, and d_18:1/24:1) at the same time points (Table 1); that is, there were no significant changes in ceramide levels in BAL at 4 and 8 h, only at 24 h. To determine whether the observed changes in SPT protein concentration reflected a change in transcription, we assessed the concentration of SPTLC1 and SPTLC2 mRNA in mouse lungs using qRT-PCR. There were no significant changes in SPTLC1 or SPTLC2 mRNA expression detectable in mouse lungs at 4, 8, or 24 h following the final NE exposure, compared with controls (data not shown). Together, these data indicate that increased SPTLC2 expression coincided with increased BAL ceramide levels observed in our model of NE-induced inflammation.

Fig. 4.

Western analyses of SPTLC1 and SPTLC2 in lungs of mice exposed to control vehicle or NE. Representative Western blots of SPT long-chain subunits 1 and 2 in mouse lung lysates at 24 h post-NE or control vehicle, probed for SPTLC1 (A and B), or SPTLC2 (C and D) after the final oropharyngeal aspiration of vehicle control or NE. In A and C, top, representative Westerns for serine palmitoyltransferase (SPT) long-chain subunits 1 or 2 (SPTLC1 or SPTLC2) and actin are shown. In B and D, bottom, relative densitometry of bands are presented following normalization to actin, shown as percentage of average control. Data are expressed as means ± SE; summarized from 2 experiments, n = 10 mice per group, and compared by Mann-Whitney U- (Wilcoxon rank sum) test; ***P < 0.001; *P < 0.05.

ORMDL3 and RTN4 inhibit SPT activity (6, 7), but their effects on the expression of SPT are unknown. Thus, an increase in SPT expression could be accompanied by a change in the levels of ORMDL3 or RTN4. Analysis of mouse whole lung lysates revealed that protein expression of ORMDL3 and RTN4 was unchanged in NE-treated mice compared with controls at 24 h (data not shown). Currently, we have no evidence to support a role for ORMDL3 or RTN4 to regulate SPT activity or expression in this mouse model. Thus, the increase in long-chain ceramide moieties (Fig. 3) was likely due to increased SPT enzymatic activity due to increased expression of SPTLC2.

Myriocin, an SPT inhibitor, decreased NE-induced ceramide.

To determine whether the increase in BAL ceramide after NE aspiration was related to an increase in de novo sphingolipid synthesis, we used a specific inhibitor of SPT activity. Myriocin, a fungus-derived suicide inhibitor, forms covalent bonds at the active site of SPT and inactivates the enzyme (45). Treatment with myriocin (0.3 mg/kg) blocked NE-induced increases in ceramide d_18:1/22:0 (P = 0.0049) and d_18:1/24:1 (P = 0.0107), and there were trends toward decreased levels of d_18:1/24:0 (P = 0.0771) as well as total ceramide (P = 0.1112) (Fig. 5). Pretreatment with myriocin did not affect the level of SPTLC2 protein in mouse lung lysates (data not shown). Thus, ceramides generated via the de novo pathway significantly contributed to the increased ceramide levels observed in BAL after NE exposure.

Fig. 5.

BAL sphingolipid analysis following myriocin pretreatment followed by NE or control vehicle. Mice were exposed to control vehicle or myriocin (0.3 mg/kg ip) 2 h before NE or control vehicle; BAL and mouse lungs were harvested on day 8. Sphingolipid concentration in BAL (200 µl) was determined by HPLC-ESI-MS/MS. Expression of total ceramides (A) and ceramide d_18:1/22:0 (B), d_18:1/24:1 (C), and d_18:1/24:0 (D) in BAL were increased following NE exposure. With the addition of myriocin, a significant decrease in d_18:1/22:0 and d_18:1/24:1 ceramide was observed, with nonsignificant decreases for the d_18:1/24:0 chain length and total ceramide. Data were normalized to the average control and compared using nonparametric one-way ANOVA (Kruskal-Wallis), with post hoc analyses among treatment groups using Mann-Whitney U- (Wilcoxon rank sum) test. Data represent the sum of 3 experiments expressed as means ± SE, with n = 15 animals per group except NE+myriocin (n = 14); ***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant, P > 0.05.

Myriocin alleviated aspects of NE-induced inflammation.

To determine whether inhibition of SPT activity by myriocin affects NE-induced inflammation, we assessed the levels of inflammatory markers present in murine BAL. Administering myriocin (0.3 mg/kg) to mice before aspiration of NE induced a significant decrease in KC (P = 0.0212) and HMGB1 (P = 0.0146) (Fig. 6). However, myriocin decreased neither the total leukocytes (total white blood cell count, total cell count) present in murine BAL nor the proportion of neutrophils present (Fig. 7). Thus, de novo ceramide regulates part, but not all, of the inflammatory effects associated with NE exposure.

Fig. 6.

Effect of myriocin on NE-induced BAL keratinocyte chemokine (KC), and high-mobility group box 1 (HMGB1). A: administration of myriocin (0.3 mg/kg) before oropharyngeal aspiration of NE decreased the level of BAL KC compared with mice that received control vehicle before NE treatment, as determined by ELISA (n = 13 per group in control, myriocin alone and NE+myriocin, n = 14 in NE). B: NE-induced expression of HMGB1 in BAL was decreased with myriocin administration (n = 13 NE and n = 12 NE+myriocin) a representative blot, and relative densitometry, normalized to HMGB1 expression against average expression with NE (shown as percentage of NE). Data summarized are means ± SE of 3 experiments, with groups compared by Mann-Whitney U- (Wilcoxon rank sum) test; ***P < 0.001; ⁺P = 0.021; *P = 0.014.

Fig. 7.

Total cell counts and percent neutrophils in BAL following pretreatment with myriocin or control vehicle and aspiration of NE or control vehicle. Aspiration of NE induced an increase in BAL total leukocytes (total cell count; A), and in percent neutrophils (B). Administration of myriocin (0.3 mg/kg ip) before NE administration did not change total cell count (total leukocytes) in murine BAL (A) or the percent neutrophils present in the BAL (B) compared with mice that received a vehicle control ip injection. Groups were compared by nonparametric ANOVA test (Kruskal-Wallis), with post hoc comparisons among groups using Mann-Whitney U- (Wilcoxon rank sum) test; data are summarized from 3 experiments as means ± SE, with n = 15 per group (vehicle control, myriocin alone, NE alone), n = 14 in NE+myriocin group; ***P < 0.001.

DISCUSSION

We have presented a novel observation: that administering exogenous NE, a mediator of neutrophilic inflammation, increased the levels of total ceramide in murine BAL, predominantly due to increases in d_18:1/22:0, d_18:1/24:0, and d_18:1/24:1 long-chain ceramide moieties. Ceramides have a broad repertoire of proinflammatory activities, which could be activated by a NE-mediated increase in ceramide accumulation. We propose that there exists a feed-forward mechanism, where NE increases production of ceramide, which may further exacerbate an inflammatory response.

Alterations in sphingolipid metabolism, particularly increased generation of ceramide, have previously been linked with pulmonary inflammation (13). Sphingolipids are multifunctional, and there is mounting evidence that indicates that the properties of ceramide are dependent on moiety chain length (17). Moreover, sphingolipid homeostasis is complex; in Cftr-deficient mouse models, ceramide levels may need to be maintained within a “normal” range for optimal cellular function (15, 18). An increase in the concentration of intracellular ceramide appears to induce inflammatory signaling and apoptosis, as well as increased epithelial permeability (29). Additionally, as the concentration of intracellular ceramide rises, more ceramide is incorporated into plasma cell membranes (4, 40). Ceramide-rich lipid rafts form and recruit components of the inflammasome, including adapter protein apoptosis-associated speck-like protein and caspase-1, to the plasma membrane (14). The increase in plasma membrane ceramide also results in abnormal distribution and more rapid turnover of proteins that are components of epithelial tight junctions: ZO-1, ZO-2, and occludin (14). Finally, a cumulative increase in pulmonary ceramide impairs innate immune function and clearance of bacteria (40).

In this report, we demonstrate that NE increased SPTLC2 protein levels in mouse lungs. The increase in airway ceramides was likely due, at least in part, to increased de novo synthesis, catalyzed by a 2.5-fold increase in expression of SPTLC2 protein. It has been postulated that SPT activity could be affected by the ratio of SPTLC2 to SPTLC3, with SPTLC2 responsible for the production of d_18:1/18:0 and long-chain ceramides, including d_18:1/22:0, d_18:1/24:0, and d_18:1/24:1 (22), and SPTLC3 responsible for the production of shorter-chain ceramides like d_18:1/14:0 and d_18:1/16:0 (19). Our results are consistent with increased SPTLC2 protein and activity. In addition, generation of long-chain ceramides may be due to increased expression of specific ceramide synthases. Ceramide synthases (CerS)2 and -5 are highly expressed in the lung and CerS2 regulates the generation of d_18:1/22:0, d_18:1/24:1, and d_18:1/24:0 ceramides (30). We plan to evaluate whether NE alters CerS expression leading to an increase in longer chain length ceramides in mouse lungs.

Regulation of SPT expression and modulators of SPT activity are poorly understood (6, 7, 12, 28). Our results suggest that NE upregulates SPTLC2 by a posttranscriptional mechanism. One mechanism that could increase SPT protein is downregulation of microRNA miR-137, 181c, −9, or 29a/b, which would permit increased transcription and/or translation of SPT (12). In future studies, we plan to evaluate whether NE exposure downregulates this panel of microRNA as one of the mechanisms to increase SPTLC2 protein levels. To the extent of our knowledge, there are two proteins known to be negative regulators of SPT activity: ORMDL3 (6, 28) and RTN4 (7), but neither has been reported to alter SPT protein expression. We measured the expression of ORMDL3 in our model of NE-induced inflammation and detected no difference in mRNA or protein in mouse lung lysates when comparing animals that were administered a vehicle control vs. NE. There are multiple studies that describe RTN4 as an inhibitor of SPT in the endothelium (7, 36). Recently RTN4 was reported to be strongly expressed in airway epithelium (47); therefore, we assessed RTN4 expression in our model. Similar to ORMDL3, the levels of RTN4 protein in mouse lungs were unchanged in our model of NE-induced inflammation.

When we inhibited SPT activity using myriocin, we observed decreased levels of specific ceramide moieties as well as a coinciding reduction in the concentration of proinflammatory signaling molecules in murine BAL, including KC. However, neither the total number of leukocytes nor the proportion of neutrophils present in the BAL decreased when mice were administered an SPT inhibitor. These data corroborate the fact that NE can activate several signaling pathways that perpetuate neutrophil-dominant airway inflammation (43). While ceramide may provide a link between NE and cytokine expression, inhibiting de novo ceramide synthesis does not abrogate all of the proinflammatory effects stimulated by NE, as evidenced by the fact that neutrophil counts did not decrease in animals treated with an SPT inhibitor. Interestingly, this apparent disconnect between KC levels and neutrophil cell counts has been observed previously. In a model of myocardial infarction, suppression of KC using neutralizing antibodies did not decrease the influx of neutrophil that occurs shortly after an infarct is induced (27). Thus, it is possible that modifying ceramide levels alone is beneficial but not sufficient to block all NE-triggered proinflammatory pathways that facilitate neutrophilic inflammation.

The above-mentioned premise does not discount the possibility that inhibition of both de novo-derived ceramides and SMase-derived ceramides will act in an additive or synergistic fashion to mitigate neutrophilic inflammation. Indeed, our study is limited by the fact that we chose to focus on de novo sphingolipid biosynthesis. We have yet to evaluate whether NE upregulates SMases or components of the salvage pathway in vivo to increase ceramide accumulation; these studies are currently in progress. For example, altered SMase-derived ceramide in the lungs of Cftr-deficient mice is attributed wholly to the loss of functioning CFTR protein (4, 40). There are several mouse models with different Cftr mutations which display varying levels of deficiency in CFTR function (48). For example, Teichgraeber et al. tested two strains of Cftr- deficient, gut-corrected mice (Cftr^tm1Unc-Tg^(FABPCFTR) and B6.129P2(CF/3)-Cftr^{TgH(neoim)Hgu}) that do not require a specialized diet; both have increased ceramide in murine airways, and in both strains of mice, CFTR dysfunction appears to result in increased acid SMase activity, thereby increasing ceramide production (40). Ceramide accumulation and inflammation in gut-corrected Cftr-deficient mice can be abrogated by decreasing the activity of acid SMase, whether by knocking down expression of the protein though genetic manipulation or through the use of SMase inhibitors (40). Likewise, two early phase II clinical trials revealed that treating patients with CF with amitriptyline (1, 25, 34), an acid SMase inhibitor, slightly increases lung function (FEV1% predicted). These trials had limited numbers of subjects and await further confirmation in larger prospective trials.

Importantly, the association between NE and ceramide may also be present in patients with CF and COPD, who suffer from acute and chronic neutrophilic inflammation with high levels of NE in airway surface fluid. Therefore, in addition to loss of CFTR function, high levels of NE may contribute to the increased ceramide detected in sputum and tissues (5). On the basis of the mechanism we describe here, increased airway ceramides may be due to NE-induced increased expression of SPTLC2 in airways of patients with chronic inflammatory lung diseases, like CF and COPD. The novel association between NE, increased SPTLC2 protein levels, de novo synthesis of ceramides, and increased expression of KC and HMGB1 in the airways is likely to alter sphingolipid metabolism in the lungs of patients with neutrophil-dominant airway inflammation. Understanding the pathways that contribute to the increased levels of ceramide in the airways of these patients may spur the development of innovative therapeutics that target the enzymes responsible for ceramide production to ameliorate chronic lung inflammation.

GRANTS

This work was supported by Virginia Commonwealth Health Research Board Grant 236-14-14 (to J. A. Voynow), CF Foundation, VOYNOW15IO (to J. A. Voynow), and Children’s Hospital Foundation Research Fund (to S. Karandashova). This work was also supported by Veteran’s Administration Merit Review Grant I BX001792 (to C. E. Chalfant), Research Career Scientist Award 13F-RCS-002 (to C. E. Chalfant)], and National Institutes of Health Grants HL-125353 (to C. E. Chalfant), HD-087198 (to C. E. Chalfant), RR-031535 (to C. E. Chalfant), and NH1-C06-RR-17393 (to Virginia Commonwealth University for renovation). Services and products in support of the research project were generated by the VCU Massey Cancer Center Shared supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059.

DISCLAIMERS

The contents of this manuscript do not represent the views of the Department of Veterans Affairs or the United States Government.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.K., C.C., and J.A.V. conceived and designed research; S.K., A.B.K., and S.Z. performed experiments; S.K., A.B.K., S.Z., and J.A.V. analyzed data; S.K., A.B.K., S.Z., C.C., and J.A.V. interpreted results of experiments; S.K. prepared figures; S.K. and J.A.V. drafted manuscript; S.K., A.B.K., S.Z., C.C., and J.A.V. edited and revised manuscript; S.K. and J.A.V. approved final version of manuscript.