Lipidomic and transcriptional analysis of the linoleoyl-omega-hydroxyceramide biosynthetic pathway in human psoriatic lesions

Figures

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  Scheme 1. Representative structures of EOx ceramides including EOS, EOH, and EOP (A) and epidermal lipoxygenase oxidation pathway (B). EOH, hydroxysphingosine; EOP, phytosphingosine; EOS, esterified omega-hydroxy sphingosine; EOx, omega-hydroxy sphingosine/phytosphingosine/hydroxysphingosine.

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  Scheme 2. Pathway diagram showing the formation and oxidation of long-chain EOS species in human skin. The same enzymes will be responsible for metabolizing EOH and EOP isomers. Gene names in red are all significantly elevated in human psoriatic lesions. EOH, hydroxysphingosine; EOP, phytosphingosine; EOS, esterified omega-hydroxy sphingosine.

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  Fig. 1. Precursor LC/MS/MS identification of native C32-EOx in the healthy human skin. Human epidermal breast tissue was prepared and analyzed using LC/MS/MS as described in Materials and methods. A: Precursor scan for m/z 279.2 ([M-H]− LA) shows a series of ions eluting between 20 and 55 min run, labeled 1–13. These are identified EOS, EOH, and EOP ceramides with varying very-long-chain fatty acid carbon numbers. B–D: Representative chromatograms and MS/MS spectra of C32 EOS, EOH, and EOP detected as the precursor ([M-H+CH3CO2H]−) → m/z 279.2. MS/MS spectra were acquired in the ion trap mode at the apex of elution for each lipid as described in Materials and methods. EOH, hydroxysphingosine; EOP, phytosphingosine; EOS, esterified omega-hydroxy sphingosine; EOx, omega-hydroxy sphingosine/phytosphingosine/hydroxysphingosine.

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  Fig. 2. Precursor LC/MS/MS identification of epoxy-HOME EOx in the human skin. Human epidermal breast tissue was prepared and analyzed as described in Materials and methods. A: Precursor scan for m/z 311.2 ([M-H]− for epoxy-HOME) shows a series of ions eluting between 14 and 22 min, labeled 1–9. These are identified as EOS, EOH, and EOP epoxy-HOME ceramides with varying very-long-chain fatty acid carbon numbers. B–D: Representative chromatograms and MS/MS spectra of C32 EOS, EOH, and EOP epoxy-HOME ceramides detected as the precursor ([M-H+CH3CO2H]−) → 311.2. MS/MS spectra were acquired in the ion trap mode at the apex of elution for each lipid as described. EOH, hydroxysphingosine; EOP, phytosphingosine; EOS, esterified omega-hydroxy sphingosine; EOx, omega-hydroxy sphingosine/phytosphingosine/hydroxysphingosine.

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  Fig. 3. Precursor LC/MS/MS identification of HODE EOx in the human skin. Human epidermal breast tissue was prepared and analyzed as described in Materials and methods. A: Precursor scan for m/z 295.2 ([M-H]− for HODE) shows a series of ions eluting between 15 and 30 min, labeled 1–9. These are identified as EOS, EOH, and EOP epoxy-HODE ceramides with varying very-long-chain fatty acid carbon numbers. B–D: Representative chromatograms and MS/MS spectra of C32 EOS, EOH, and EOP HODE ceramides detected as precursor ([M-H+CH3CO2H]−) → 295.2. MS/MS spectra were acquired in the ion trap mode at the apex of elution for each lipid as described. EOH, hydroxysphingosine; EOP, phytosphingosine; EOS, esterified omega-hydroxy sphingosine; EOx, omega-hydroxy sphingosine/phytosphingosine/hydroxysphingosine.

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  Fig. 4. Precursor LC/MS/MS identification of triHOME EOx in the human skin. Human epidermal breast tissue was prepared and analyzed as described in Materials and methods. A: Precursor scan for m/z 329.2 ([M-H]− for triHOME) shows a series of ions eluting between 12 and 18 min, labeled 1–9. These are identified as EOS, EOH, and EOP triHOME ceramides with varying very-long-chain fatty acid carbon numbers. B–D: Representative chromatograms and MS/MS spectra of C32 EOS, EOH, and EOP triHOME ceramides detected as the precursor ([M-H+CH3CO2H]−) → 329.2. MS/MS spectra were acquired in the ion trap mode at the apex of elution for each lipid as described. EOH, hydroxysphingosine; EOP, phytosphingosine; EOS, esterified omega-hydroxy sphingosine; EOx, omega-hydroxy sphingosine/phytosphingosine/hydroxysphingosine.

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  Fig. 5. Analysis of triHOME, HODE, and epoxy-HOME EOx ceramides and free oxylipins in the healthy human epidermis. All lipids were extracted and analyzed using LC/MS/MS from human epidermal breast tissue as described in Materials and methods (n = 9, mean ± SEM). A: LA-EOx-LA ceramide lipids in the healthy epidermis. B–D: Epidermis levels of EOx oxidized ceramides in the healthy human epidermis. E–H: Free oxylipin quantification in the healthy epidermis. LA, linoleic acid; EOx, omega-hydroxy sphingosine/phytosphingosine/hydroxysphingosine.

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  Fig. 6. Human epidermis shows higher levels of triHOME and HODE-EOS and reduced levels of epoxyHOME-EOS in upper layers. Tape strips were acquired from the volar forearm of healthy volunteers (n = 5 subjects, 30 strips per person, mean ± SEM). Strips were combined into groups of 3 and lipids extracted and analyzed using LC/MS/MS as described in Materials and methods for total levels of lipid present. A: LA-EOS is evenly distributed throughout the skin layers. B: Epoxy-HOME EOS increases with greater epidermal depth and is the highest in the stratum granulosum (SG)/stratum spinosum (SS) layers. C and D: TriHOME EOS and HODE EOS are increased in the upper layers of the epidermis and appear the highest in the stratum corneum (SC). EOS, esterified omega-hydroxy sphingosine; LA, linoleic acid.

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  Fig. 7. Lipidomics analysis of psoriasis lesions shows elevated native EOS species and significantly altered levels of oxidized species. Tape strips were acquired from the volar forearm (uninvolved skin) and elbow (location of psoriatic lesion), from patients with psoriasis (n = 9) and from healthy controls (n = 5). From each site, 9 tape strips were obtained and pooled into groups of 3. Lipids were extracted and analyzed as described in Materials and methods. Increasing the tape strip number corresponds to increasing epidermal depth. Psoriatic patients (empty bars), healthy controls (gray bars), mean ± SEM.∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001. The bold down arrow (↓) corresponds to skin lesion samples. Geometric shapes show outliers. A: Epidermal tape strip profile of C30 and C32 EOS. B: Epidermal tape strip profile of triHOME EOS. C: Epidermal tape strip profile of 9-HODE EOS in psoriatic versus healthy control. EOS, esterified omega-hydroxy sphingosine.

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  Fig. 8. Free triHOMEs and HODEs are elevated in human psoriatic lesions. Tape strips were acquired from the volar forearm (uninvolved skin) and elbow (location of psoriatic lesion), from patients with psoriasis (n = 9) and healthy controls (n = 5). From each site, nine tape strips were obtained and pooled into groups of 3. Lipids were extracted and analyzed as described in Materials and methods. An increasing tape strip number corresponds to increasing epidermal depth. Psoriatic patients (empty bars), healthy controls (gray bars), mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. The bold down arrow (↓) corresponds to skin lesion samples. Geometric shapes show outliers. A: 9-HODE. B: 13-HODE. C: 9,10,13 triHOME. D: 9,12,13 triHOME.

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  Fig. 9. Several ceramide pathway genes are upregulated in psoriasis and functionally linked with the transcription factor ZIC1. Affymetrix CEL files from a psoriasis study were downloaded and genes in the ceramide pathway (Scheme 2) analyzed (17). Robust multiarray averaging-normalized gene expression level was corrected for multiple comparisons on a total of 50,683 probes (Benjamini-Hochberg test). Psoriasis plaque (PP), n = 64; psoriasis normal skin (PN), n = 58; healthy control normal skin (NN), n = 58. A: Box and whisker plots showing gene expression level for genes in the ceramide pathway which are significantly different. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.005. B: IPA identifies many significantly different genes in the ceramide pathway link to ZIC1. Gene identifiers and expression values were uploaded for significant genes and network analysis performed (adjusted P value < 0.01). IPA, Ingenuity Pathway Analysis.

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  Fig. 10. Expression of phospholipases, ALOX15B, and TGM genes in psoriasis shows candidate PLA2 genes for oxylipin hydrolysis from ceramides and significant elevation in TGMs in psoriasis lesions. Affymetrix CEL files from a psoriasis study were downloaded and genes in the ceramide pathway analyzed (17). RMA-normalized gene expression level was corrected for multiple comparisons on a total of 50,683 probes (Benjamini-Hochberg test). Psoriasis plaque (PP), n = 64; psoriasis normal skin (PN), n = 58; healthy control normal skin (NN) n = 58. Box and whisker plots show gene expression level for genes in the ceramide pathway, which are significantly different. ∗∗P < 0.01, ∗∗∗P < 0.005.