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Methods

Utilizing myriocin, a potent inhibitor of sphingolipid synthesis, we evaluated the immune regulatory role of de novo ceramide generation in vitro and in vivo. Intratracheal myriocin was administered alone or during house dust mite sensitization (HDM) of BALB/C mice and airway hyper‐responsiveness (AHR) was evaluated by invasive plethysmography followed by bronchial lavage (BAL) cytology and cytokine quantification.

Results

Myriocin inhibits and HDM exposure activates de novo ceramide synthesis in bone marrow‐derived dendritic cells. Mice receiving intratracheal myriocin developed a mild airway neutrophilic infiltrate without inducing a significant increase in AHR. CXCL1 was elevated in the BAL fluid of myriocin‐treated mice while the neutrophilic chemotactic factors anaphylatoxin C5a, leukotriene B4, and IL‐17 were unaffected. HDM treatment combined with myriocin led to a dramatic enhancement of AHR (63% increase over HDM alone, p < 0.001) and increased granulocyte pulmonary infiltrates versus HDM or myriocin alone. Elevated Th2 T cell counts and Th2 cytokines/chemokines (IL5, IL13, CCL17) were observed in mice treated with combined HDM/myriocin compared to HDM alone. Myriocin‐treated pulmonary CD11c+ cells stimulated with HDM secreted significantly more CXCL1 than cells stimulated with HDM alone while HDM stimulated airway epithelial cells showed no change in CXCL1 secretion following myriocin treatment.

Animals, immunizations, and materials

Seven‐ to eight‐week‐old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME), maintained in a specific pathogen free facility at Cincinnati Children's Hospital Medical Center (CCHMC) and handled under Institutional Animal Care and Use Committee‐approved procedures. Following isoflourane anesthesia, animals underwent allergen immunization as previously described with the following modifications: mice were serially immunized intratracheally every 48 h for 2 weeks with low‐endotoxin, low‐dose HDM extract (D. pteronyssinus, Greer Labs; Lenoir, NC) (3 μg total protein/dose in 50 μL of sterile phosphate‐buffered saline plus vehicle [PBS]). Myriocin (Cayman Chemical Company; Ann Arbor, MI) stock solutions (5 mM) were prepared in methanol, and aqueous solutions were prepared fresh before each experiment (1.2 μg/intratracheal dose). Ceramide C6 (N‐hexanoyl‐D‐erythro‐sphingosine) was obtained from Avanti Polar Lipids (Alabaster, AL), mouse GM‐CSF was acquired from PeproTech(Rocky Hill, NJ), and RPMI and serine‐free MEM were purchased from Gibco (Carsbad, CA). Normal human bronchial epithelial cells (NHBE) were obtained from Lonza (Walkerville, MD) and cultured per the manufacturer's instructions. Murine bone marrow‐derived dendritic cells (BMDCs) were generated as previously described .

Pulmonary SPT inhibition enhances airway hyper‐responsiveness

Allergen‐induced AHR is a robust physiologic end‐point for quantitating the influence of bioactive compounds on the pathophysiology of allergic asthma. Using a well‐characterized murine model of HDM‐induced AHR, we investigated the biochemical and physiologic effects of intratracheal myriocin on lung lipid content, lung function, and airway structure (treatment protocol outlined in Fig. A). Similar to other investigators, we administered a low dose of intratracheal HDM extract, which sub‐maximally induces AHR and airway inflammation, thus increasing our ability to detect additive and synergistic effects , , , . First, we performed AHR measurement on mice after eight serial doses of intratracheal myriocin and did not detect a significant rise in AHR compared with vehicle‐treated control mice (Fig. B) despite a sustained reduction in lung ceramide levels (Fig. C). Next, we performed histologic examination of lung tissue from myriocin‐treated mice, which showed normal bronchial and alveolar architecture, similar to vehicle‐treated mice (Fig. A vs. D).

Pulmonary SPT inhibition enhances airway hyper‐responsiveness

After establishing that intratracheal myriocin does not induce significant AHR in our system, we compared the effect of HDM/myriocin co‐administration with that of HDM alone and found that co‐administration significantly enhanced AHR (p < 0.001) (Fig. B). Myriocin's effect on lung ceramide levels, however, was undetectable in animals co‐treated with HDM/myriocin (Fig. C,Supplemental Fig. S1B). Lung histology from both HDM only and HDM/myriocin‐treated mice revealed similar levels of peribronchial inflammatory infiltrates (Fig. B vs. E) and goblet cell metaplasia (Fig. C vs. F, Supplementary Fig. S1C). Goblet cell gene expression analysis revealed that HDM exposure induced multiple asthma‐associated transcripts, including chloride channel calcium‐activated 1 (Clca1/Gob‐5), mucin 5, subtypes A and C, tracheobronchial/gastric (Muc5ac), intelectin 1 (Itln1), and intelectin 2 (Itln2/B). Compared to HDM alone, HDM/myriocin co‐administration enhanced Itln1 and Itln2 expression, had no effect on Clca1, and significantly decreased Muc5ac expression. Therefore, in an allergic model of asthma, myriocin‐treatment is associated with enhanced HDM‐induced AHR and an altered goblet cell gene expression pattern.

Results

This validated HPLC-LTQ-Orbitrap-HRMS method was used for the quantification analysis of myriocin in 7 batches of Cordyceps under the SRM mode. The negative ion spectra shows intense signal at m/z 400.2646 for myriocin. The main fragment in the low m/z spectral region derive from the threonine portion (formation of deprotonated serine, m/z 104.0333, C3H6NO3+), and fission of the bonds close to the C-14 carbonyl group yields a minor fragment at m/z 85.0226, C6H13+). The first-generation loss of the elements of formaldehyde (CH2O; −30 Da) and subsequent loss of two water molecules (H2O, H2O; −18–18 Da) were yielded from the precursor ion [M-H]− (m/z 400.2646) to derive the fragments at m/z 370.4453, m/z 352.2433 and m/z 334.2335, respectively, as shown in . The content of myriocin was calculated by its calibration curve, and the quantification results were presented in and . By comparison of the retention time and fragmentation pattern with a reference standard, myriocin was identified and confirmed, for the target peak showed the same retention time at 11.20 and the same fragmentation as the standard (). As presented in , myriocin was quantified in sample D, C and B at the levels of 189.36, 49.49, 9.80 μg/g, respectively. But it was very low and under the limit of determination in sample E, F, G and H, which were classified as Isaria cicadae.10.1080/21501203.2017.1383319-F0002Figure 2.Ion map(SIM)of vegetable cicada and its allies. (a): Myriocin (1: 11.20 min); (b): Cordyceps cicadae; (c): Fruiting body of Ophiocordyceps longissima; (d): Mycelium of O. longissima; (e): Fruiting body of vegetable cicada; (f): Substrate of vegetable cicada; (g): Wild vegetable cicada (Anhui); (h): Wild vegetable cicada (Henan).10.1080/21501203.2017.1383319-T0001Table 1.The content of myriocin in vegetable cicada and its allies.SampleOriginSourceContent(μg/g)BWild Cordyceps cicadaeYunnan9.80CFruiting body of O. longissimaAnhui Agricultural University49.49DMycelium of O. longissimaAnhui Agricultural University189.36EFruiting body of vegetable cicadaZhejiang Bioasia Institute of Life SciencesUDFSubstrate of vegetable cicadaZhejiang Bioasia Institute of Life SciencesUDGWild vegetable cicadaAnhuiUDHWild vegetable cicadaHenanUDUD: under the limit of determination.

Abstract

Myriocin is an antibiotic derived from Mycelia sterilia, and is a potent inhibitor of serine palmitoyltransferase, the enzyme involved in the first step of sphingosine synthesis. Myriocin, inhibiting ceramide synthesis, has a great potential for treatment of diseases characterized by high ceramide levels in affected tissues, such as retinitis pigmentosa (RP). Drug delivery to the retina is a challenging task, which is generally by-passed through intravitreal injection, that represents a risky invasive procedure. We, therefore, developed and characterized an ophthalmic topical nanotechnological formulation based on a nanostructured lipid carrier (NLC) and containing myriocin. The ocular distribution of myriocin in the back of the eye was assessed both in rabbits and mice using LC-MS/MS. Moreover, rabbit retinal sphingolipid and ceramides levels, after myriocin-NLC (Myr-NLC) eye drops treatment, were assessed. The results demonstrated that Myr-NLC formulation is well tolerated and provided effective levels of myriocin in the back of the eye both in rabbits and mice. We found that Myr-NLC eye drops treatment was able to significantly decrease retinal sphingolipid levels. In conclusion, these data suggest that the Myr-NLC ophthalmic formulation is suitable for pharmaceutical development and warrants further clinical evaluation of this eye drops for the treatment of RP.

Ocular pharmacokinetics and tolerability

Our experimental protocol complied with the statements of the Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and visual research. New Zealand (NZ) albino rabbits (weight 2.0–2.5 kg) were purchased from Envigo s.r.l (San Piero al Natisone, Udine, Italy). Rabbit can be considered a reliable animal model for translational ocular pharmacokinetics studies (Durairaj, ). Rabbits were housed in standard conditions for one week before the experiment (light-controlled room, controlled temperature 22 ± 1 °C and humidity 10–30%). Rabbits were randomly assigned to two experimental groups (treated and vehicle): n = 10 per experimental group, 2 rabbits and 4 retinas per time point. The two experimental groups of rabbits received ocular topical administration, 30 µl × 2 times within 5 min of the interval, of Myr-NLC (NLC1, drug loading 0.68 mg/ml) and vehicle (unloaded NLC), respectively. Rabbits were sacrificed at 30, 60, 120, 180, 240 min after ocular topical administration of the myriocin-NLC and unloaded NLC. Rabbits were sacrificed by intravenous administration of 0.3 ml/kg of Tanax© (Intervet, Milano, Italy), after sedation with an intramuscular administration of 10 mg/kg of Zoletil© (Virbac, Milano, Italy). Eyes were enucleated and ocular tissues (vitreous and retina) were collected. Tissue samples were stored at −80 C° until quantitative analysis of myriocin. Ocular distribution of myriocin was determined also in the retina of C57BL6J mice after topical administration of myriocin-NLC formulation (NLC1, drug loading 0.68 mg/ml). In order to increase formulation corneal residency, three 5 µl administrations within 2 min of interval were carried out. The amount of topical administered myriocin was 15 µg. C57BL6J mice (n = 4) per group were sacrificed 30, 60, 180 and 240 min after ocular topical administration of myriocin-NLC formulation; a separate set of animals (n = 12) was used to determine myriocin in control mice (vehicle). Mice were sacrificed by cervical dislocation; the eye enucleated and retina collected and stored at −80 °C until analysis.

Ocular pharmacokinetics and tolerability

The following pharmacokinetics parameters were determined in rabbit and mouse eye tissues: peak eye tissue concentration (Cmax), and time of peak of eye tissue concentration (Tmax). The area under the curve (AUC0-240) of eye tissues was extrapolated from the concentration of myriocin [myriocin] vs. time curves. Ocular safety of unloaded NLC and Myr-NLC formulations were evaluated by a modified Draize’s test in a separate set of rabbits (n = 6) (Leonardi et al., ; Giannavola et al., ). Formulations were topically administered (30 μl) in the eye every 30 min for 6 h (12 treatments). Scores were assigned accordingly to previous studies, and eyes were examined at 10 min and 6 h after Myr-NLC administration by a slit lamp (4179 T Sbisà, Florence, Italy). Observations were made by two independent observers in a masked way.

Ocular pharmacokinetics and tolerability

Our experimental protocol complied with the statements of the Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and visual research. New Zealand (NZ) albino rabbits (weight 2.0–2.5 kg) were purchased from Envigo s.r.l (San Piero al Natisone, Udine, Italy). Rabbit can be considered a reliable animal model for translational ocular pharmacokinetics studies (Durairaj, ). Rabbits were housed in standard conditions for one week before the experiment (light-controlled room, controlled temperature 22 ± 1 °C and humidity 10–30%). Rabbits were randomly assigned to two experimental groups (treated and vehicle): n = 10 per experimental group, 2 rabbits and 4 retinas per time point. The two experimental groups of rabbits received ocular topical administration, 30 µl × 2 times within 5 min of the interval, of Myr-NLC (NLC1, drug loading 0.68 mg/ml) and vehicle (unloaded NLC), respectively. Rabbits were sacrificed at 30, 60, 120, 180, 240 min after ocular topical administration of the myriocin-NLC and unloaded NLC. Rabbits were sacrificed by intravenous administration of 0.3 ml/kg of Tanax© (Intervet, Milano, Italy), after sedation with an intramuscular administration of 10 mg/kg of Zoletil© (Virbac, Milano, Italy). Eyes were enucleated and ocular tissues (vitreous and retina) were collected. Tissue samples were stored at −80 C° until quantitative analysis of myriocin. Ocular distribution of myriocin was determined also in the retina of C57BL6J mice after topical administration of myriocin-NLC formulation (NLC1, drug loading 0.68 mg/ml). In order to increase formulation corneal residency, three 5 µl administrations within 2 min of interval were carried out. The amount of topical administered myriocin was 15 µg. C57BL6J mice (n = 4) per group were sacrificed 30, 60, 180 and 240 min after ocular topical administration of myriocin-NLC formulation; a separate set of animals (n = 12) was used to determine myriocin in control mice (vehicle). Mice were sacrificed by cervical dislocation; the eye enucleated and retina collected and stored at −80 °C until analysis.

Abstract

The modification of lipid composition allows cells to adjust membrane biophysical properties in response to changes in environmental temperature. Here, we use adaptive laboratory evolution (ALE) in the presence of myriocin, a sphingolipid (SLs) biosynthesis inhibitor, to remodel the lipid profile of an industrial yeast strain (LH) of Saccharomyces cerevisiae. The approach enabled to obtain a heterogeneous population (LHev) of myriocin‐tolerant evolved clones characterized by its growth capacity at high temperature. Myriocin exposure also caused tolerance to soraphen A, an inhibitor of the acetyl‐CoA carboxylase Acc1, the rate‐limiting enzyme in fatty acid de novo production, supporting a change in lipid metabolism during ALE. In line with this, characterization of two randomly selected clones, LH03 and LH09, showed the presence of lipids with increased saturation degree and reduced acyl length. In addition, the clone LH03, which displays the greater improvement in fitness at 40°C, exhibited higher SL content as compared with the parental strain. Analysis of the LH03 and LH09 genomes revealed a loss of chromosomes affecting genes that have a role in fatty acid synthesis and elongation. The link between ploidy level and growth at high temperature was further supported by the analysis of a fully isogenic set of yeast strains with ploidy between 1N and 4N which showed that the loss of genome content provides heat tolerance. Consistent with this, a thermotolerant evolved population (LH40°) generated from the parental LH strain by heat‐driven ALE exhibited a reduction in the chromosome copy number. Thus, our results identify myriocin‐driven evolution as a powerful approach to investigate the mechanisms of acquired thermotolerance and to generate improved strains.

Adaptive evolution in the presence of myriocin

We used a robust industrial baker's yeast strain named L'Hirondelle (LH) in order to force by ALE changes in the lipid metabolism. For this, yeast was propagated by successive batch refreshments maintained constantly in the presence of myriocin at 30°C during 50 generations (see Fig. A). In these experiments, a chronic‐medium dose of 1.2 μM myriocin was used to lower but not completely inhibit SPT activity (Huang et al., ; Fig. B). Moreover, a short‐term experiment was chosen because deleterious mutations, which could affect negatively important traits of industrial strains, such as cell size, fitness or carbon‐source utilization (Aguilera et al., ; Wenger et al., ; Çakar et al., ; Strucko et al., ), have been reported to accumulate over time (Tenaillon et al., ). In general, the rates of spontaneous mutation are higher for neutral and deleterious mutations and lower for beneficial mutations (Barrick and Lenski, ; Van den Bergh et al., ). In this manner, we seek to generate evolved clones containing stable compensatory mutations conferring increased SL biosynthesis (see Fig. B) or altered PL asymmetry, but still maintaining key industrial properties. We found that the doubling time decreased gradually over the course of the experiment (data not shown), indicating that the original yeast population was capable of adapting to optimize growth in the presence of the drug. As shown in Fig. C, the 50‐generations evolved population (LHev) grew in a solid myriocin‐containing YPD medium, while the parental strain did not. As expected, tolerance to myriocin was also observed for 10 individual adaptive clones isolated from the terminal population. In addition, all of them showed a similar growth (Fig. C), suggesting that adaptation‐driving mutations were distributed across the population (Tenaillon et al., ).

Myriocin‐driven evolution alters yeast growth at different temperatures

We asked whether the changes in myriocin tolerance observed in the evolved population were relevant for the physiological response to thermal stress. As shown in Fig. , growth at 30°C, the optimal temperature for S. cerevisiae of the yeast population was unaffected by the evolutionary experiment. On the contrary, the terminal population exhibited increased growth at 40°C as compared with the parental, whereas a trade‐off in cell proliferation under cold conditions was observed (Fig. ). Likewise, most of the selected clones under study (LH01–LH10) exhibited increased fitness at 40°C (data not shown) although again the phenotype differed quantitatively among individuals as it is illustrated for two randomly selected clones, LH03 and LH09 (Fig. ). Thus, the clone LH09 exhibited only a slight growth advantage at 40°C, as compared with the parental LH strain, while the LH03 clone grew much faster and had a similar behaviour to that of the LHev population. Finally, these phenotypes correlated again with a loss in cold growth (Fig. ). We conclude that myriocin‐drive evolution improved yeasts' ability to adapt to increased temperature with no apparent trade‐offs at the ancestral optimal temperature of 30°C. The results also highlight how tolerance to high temperature is associated with fitness impairments under cold‐stressful conditions.