Lipidomic and transcriptional analysis of the Linoleoyl-omega-Hydroxyceramide biosynthetic pathway in human psoriatic lesions

 Precursor scanning of human epidermal extracts identifies a series of ceramides that contain LAHuman epidermal extracts were analysed using precursor-LC/MS/MS for the LA carboxylate anion (m/z 279.2 [M-H]-). Figure 1 A shows a series of ions eluting between 22 – 55 min. MS spectra acquired at the apex of each peak show precursor ions that correspond to putative LA-containing ceramides. This represents multiple species in the range of C28-C36 EOS, EOP and EOH, with the order of elution corresponding to lipophilicity of the ceramide functional groups and the length of the N-linked fatty acyl (Figure 11, Supplemental Figure 1). Peak 1 has been putatively assigned (based on parent mass) as C30 EOH at m/z 1086.9 [M-H+CH3CO2H]- (also detected as m/z 1026.9 [M-H]-) (see Peak 1 in Supplemental Figure 1). Peak 2 indicates the less polar (more lipophilic) C31 EOH ceramide (m/z 1100.9 [M-H+CH3CO2H]-, m/z 1040.9 [M-H]-). This pattern continues with increasing retention time through Peaks 3-13 (Supplemental Figure 1). Next, lipid extracts were analysed in MRM mode for EOS, EOH and EOP C30, C32 and C34 species, using the product ion for LA [M-H]- at m/z 279.2 and the [M-H+CH3CO2H]- precursor ion. C32 EOS, EOH and EOP species are shown in Figure 1 B, C and D respectively. Here, peaks are seen for each lipid and a representative MS/MS spectrum for each demonstrates the expected product ions including the [M-H+CH3CO2H]- acetate adduct (marked with *) and also [M-H]- (marked with +), m/z 279.2 (fatty acyl carboxylate from cleavage of the ω-hydroxylated LA (Figure 1 B-D). Supplemental Figure 2 illustrates the MS/MS of C30 EOS (Panel A), EOH (Panel B) and EOP (Panel C) ceramides. MRM chromatograms for C34 EOS (Panel A), EOH (Panel B) and EOP (Panel C) are shown in Supplemental Figure 3, with relative retention times inferring the corresponding structures as indicated. Consistent throughout all spectra are product ions corresponding to NL 262 amu, representing loss of the LA ketene moiety on CID. Up to now, most studies on skin ceramides have used positive ion mode, however since we focused mainly on oxidized forms, we needed to use negative ion mode in order to detect and characterise the esterified oxylipin groups, especially during precursor scanning which is required for this purpose. However, there is an absence of commercially available standards for these long chain acyl-esterified ceramides, so we were only able to obtain synthetic LA-C30EOS for comparison. As expected, this showed an identical retention time and MS/MS spectrum to the putative LA-C30EOS detected in skin (Supplemental Figure 4). High resolution MS/MS for the standard is also shown in negative ion mode (Supplemental Figure 4 B,C). As already seen for positive MS of skin ceramides, diagnostic ions were seen for loss of fatty acyl (NL 280 amu at m/z 748.7) and its respective ketene (NL 262 amu at m/z 718) (van Smeden J. Hoppel L. van der Heijden R. Hankemeier T. Vreeken R.J. Bouwstra J.A. LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery.).Figure thumbnail gr1

Figure 1Precursor LC/MS/MS identification of native C32-EOx in healthy human skin. Human epidermal breast tissue was prepared and analysed using LC/MS/MS as described in Methods. Panel A. Precursor scan for m/z 279.2 ([M-H]-LA) shows a series of ions eluting between 20-55 min run, labelled 1-13. These are identified EOS, EOH and EOP ceramides with varying very long chain fatty acid carbon number. Panels B-D. Representative chromatograms and MS/MS spectra of C32 EOS, EOH and EOP detected as precursor ([M-H+CH3CO2H]-) → m/z 279.2. MS/MS spectra were acquired in ion trap mode at the apex of elution for each lipid as described in Methods.

 Precursor scanning identifies a series of ceramide lipids that contain oxidised LA in human epidermal extracts

Precursor-LC/MS/MS was next used to screen for oxidised EOx putatively generated by 12R-LOX/eLOX3. The product ions used were m/z 311.2 (epoxy-HOME), 295.2 (HODE) and 329.2 (triHOME), [M-H]-. We note that these ions do not specify the position of oxygenation on LA, and furthermore don’t distinguish the precise oxygenated functional groups at this stage of the analysis.

Putative epoxy-HOME-EOS, EOP and EOH lipids eluted earlier than native precursors due to reduced lipophilicity from the epoxy-hydroxy functional group, around 14 – 22min with nine major peaks (Peaks 1-9, Figure 2 A, Supplemental Figure 5). Peak 1 with m/z 1118.9 [M-H+CH3CO2H]- was putatively assigned to the acetate adduct of epoxy-HOME EOH C30 (also seen as m/z 1058.9 [M-H]-) (Supplemental Figure 5). Peak 2, m/z 1132.9 [M-H+CH3CO2H]- was putatively assigned as epoxy-HOME EOH C31 ceramide (Supplemental Figure 5). Peaks 3-9 were then assigned based on parent mass. The chromatograms and product ion spectra for the epoxy-HOME-EOS, EOH and EOP C32 are shown (Figure 2 B-D respectively). Similar to the EOx LA substrates, each ceramide species was detected as an [M-H+CH3CO2H]- acetate adduct and also an [M-H]- ion (Figure 2 B-D). The fatty acyl product ion (the ω-hydroxylated esterified epoxy-HOME) is also consistently detected (m/z 311.2 [M-H]-). A m/z 171.2 [M-H]- product ion is also detected for all three indicating the 9,10 epoxide,13 hydroxy positional isomer. Unfortunately, there are currently no available epoxy-HOME standards with which to conduct an MS comparison. However, this finding is consistent with the positional specific oxidation reaction of the 12R-LOX enzyme which ultimately yields the 9,10,13-triHOME (Thomas C.P. Boeglin W.E. Garcia-Diaz Y. O'Donnell V.B. Brash A.R. Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis., Chiba T. Thomas C.P. Calcutt M.W. Boeglin W.E. O'Donnell V.B. Brash A.R. The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.). Supplemental Figure 6 A-C illustrates MS/MS analysis of the epoxy-HOME-EOS (Panel A), EOH (Panel B) and EOP (Panel C) C30 species and again, these show similar fragmentation. MRM chromatograms for the epoxyHOME-EOS (Panel A), EOH (Panel B) and EOP (Panel C) C34 species are shown in Supplemental Figure 7, with relative retention times inferring the corresponding structures as indicated. For EOH and EOP, the ions were lower abundance as indicated by the chromatograms. As expected, several epoxyHOME EOx species showed products ions corresponding to NL 294, representing loss of the epoxyHOME ketene moiety on CID.Figure thumbnail gr2

Figure 2Precursor LC/MS/MS identification of epoxy-HOME EOx in human skin. Human epidermal breast tissue was prepared and analysed as described in Methods. Panel A. Precursor scan for m/z 311.2 ([M-H]-for epoxy-HOME) shows a series of ions eluting between 14-22 min, labelled 1-9. These are identified as EOS, EOH and EOP epoxy-HOME ceramides with varying very long chain fatty acid carbon number. Panels B-D. Representative chromatograms and MS/MS spectra of C32 EOS, EOH and EOP epoxy-HOME ceramides detected as precursor ([M-H+CH3CO2H]-) → 311.2. MS/MS spectra were acquired in ion trap mode at the apex of elution for each lipid as described.

Precursor scanning LC/MS/MS for m/z 295.2, corresponding to HODE-EOx was undertaken and identified a series of EOS, EOP and EOH species (Figure 3 A, Supplemental Figure 8). As before, a consistent pattern in putative assignments is seen in peaks 1-9. The LC/MS/MS and product ion spectra for HODE-EOS, EOH and EOP C32 are shown (Figure 3 B-D). A m/z 171.2 [M-H]- daughter ion corresponding to fragmentation of the oxidised LA between C9-C10 is seen (consistent with 9-HODE), supporting C9 oxidation of linoleate via 12R-LOX. Supplemental Figure 9 A-C illustrates MS/MS analysis of the HODE-EOS (Panel A), EOH (Panel B) and EOP (Panel C) C30 species and again, these show daughter ion fragments corresponding to 9-HODE. The MRM chromatograms for the HODE-EOS (Panel A), EOH (Panel B) and EOP (Panel C) C34 species are shown in Supplemental Figure 10, with relative retention times inferring the corresponding structures as indicated. As expected, several HODE EOx species showed product ions corresponding to NL 278 (loss of HODE ketene) and sometimes NL 296 (loss of HODE) on CID.Figure thumbnail gr3

Figure 3Precursor LC/MS/MS identification of HODE EOx in human skin. Human epidermal breast tissue was prepared and analysed as described in Methods. Panel A. Precursor scan for m/z 295.2 ([M-H]-for HODE) shows a series of ions eluting between 15-30 min, labelled 1-9. These are identified as EOS, EOH and EOP epoxy-HODE ceramides with varying very long chain fatty acid carbon number. Panels 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 ion trap mode at the apex of elution for each lipid as described.

Putative triHOME-EOx lipids eluted earlier than the other oxidised EOx, due to a further reduction in lipophilicity from the triol functional group, around 12 – 17 minutes (Figure 4, Supplemental Figure 11). Peak 1 with m/z 1136.9 [M-H+CH3CO2H]- corresponded to the acetate adduct of triHOME EOH C30 (also seen as m/z 1076.9 [M-H]-) (Supplemental Figure 11). Peak 2 at m/z 1150.9 [M-H+CH3CO2H]- corresponds to the trihydroxy-LA C31 EOH ceramide (Supplemental Figure 11). Overall, the pattern of elution mirrors the ceramides described in Figure 1, Figure 2, Figure 3. LC/MS/MS, monitoring precursor [M-H+CH3CO2H]- to triHOME carboxylate anion corresponding to EOS, EOH and EOP C32 is shown (Figure 4 B-D). Similar to the EOx LA substrates, each ceramide species was detected as an [M-H+CH3CO2H]- acetate adduct and also an [M-H]- ion (Figure 4 B-D). The fatty acyl product ion (the ω-hydroxylated esterified trihydroxy-LA) is also consistently detected (m/z 329.2 [M-H]-). A m/z 171.2 [M-H]- product ion is seen suggesting the 9,10,13-triHOME isomer. To further confirm this, MS/MS of 9,10,13- and 9,12,13-triHOME free acids was undertaken for comparison (Supplemental Figure 11A,B). Here, m/z 171 is seen for both positional isomers, however it is very low abundance in the 9,12,13-isomer, where instead two ions at m/z 211 and 229 predominate. These ions were not detected in triHOME EOx lipids, which only contained the m/z 171 fragment. This confirms the isomer in the ceramides as the 9,10,13-form (Supplementary Figure 12) and is consistent with the positional specific oxidation reaction of the epidermal 12R-LOX enzyme which has been previously found to yield the 9,10,13 triHOME (Thomas C.P. Boeglin W.E. Garcia-Diaz Y. O'Donnell V.B. Brash A.R. Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis., Chiba T. Thomas C.P. Calcutt M.W. Boeglin W.E. O'Donnell V.B. Brash A.R. The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.). Last, many of the triHOME-EOx species yielded characteristic daughter ions for the NL of the ketene (loss of 312 amu). The presence of multiple triHOME-EOx lipids suggests that 12R-LOX and eLOX3 utilise many different epidermal ceramides to yield a large number of oxidised, highly polar products. Supplementary Figure 13 A-C illustrates MS/MS analysis of the triHOME-EOS (Panel A), EOH (Panel B) and EOP (Panel C) C30 species and again, these show daughter ion fragments (171.2) corresponding to 9,10,13 triHOME. The MRM chromatograms for the triHOME-EOS (Panel A), EOH (Panel B) and EOP (Panel C) C34 species are shown in Supplementary Figure 14.Figure thumbnail gr4

Figure 4Precursor LC/MS/MS identification of triHOME EOx in human skin. Human epidermal breast tissue was prepared and analysed as described in Methods. Panel A. Precursor scan for m/z 329.2 ([M-H]-for triHOME) shows a series of ions eluting between 12-18 min, labelled 1-9. These are identified as EOS, EOH and EOP triHOME ceramides with varying very long chain fatty acid carbon number. Panels B-D. Representative chromatograms and MS/MS spectra of C32 EOS, EOH and EOP triHOME ceramides detected as precursor ([M-H+CH3CO2H]-) → 329.2. MS/MS spectra were acquired in ion trap mode at the apex of elution for each lipid as described.

 Semi quantitation of triHOME, HODE fatty acids esterified in EOx ceramides, and free oxylipins.LA-EOS, EOH and EOP molecular species containing an ultra-long N-linked fatty acid of C30-C34 carbons were semi-quantified in healthy human epidermal tissue using LC/MS/MS. Standards for EOP and EOH are not available, and we could only obtain one C32 EOS. Thus, to allow semi-quantitation this lipid was used as primary standard. We acknowledge some limitations with this approach which include (i) longer and shorter ceramide chains may show differing ionisation efficiency, and (ii) EOS may ionise differently to EOP and EOH. We note that for all EOx analysed, the LA carboxylate anion was used as product ion, since we expect fragmentation to generate this to be similar for all lipids analysed. Of the three ceramide families, EOH appeared the most abundant followed by EOS, then EOP (Figure 5A). EOS and EOH species showed a similar relative abundance of molecular species in relation to the length of the very long N-linked carbon chain of the ceramide. Generally, the C32 was most abundant, with lower levels of C30 and C34. EOP ceramides showed similar levels of C30, C32 and C34. Since no purified triHOME-, HODE- or epoxy-HOME-containing ceramides are available, these are shown as relative to internal standard, normalised per mg tissue (Figure 5 B-D). The same relative distribution of sub-species based on carbon chain length is seen for these as for native LA-containing ceramides (Figure 5A-D). This indicates that the LOX pathway likely metabolises ceramide substrates based on their availability with no preference for any specific ceramide chain length. As a second approach to quantitation, levels of triHOME and HODEs in EOx ceramides were determined following hydrolysis of HPLC-purified EOx generated from epidermal lipid extracts. These values were next used to generate an estimate of total EOx amounts in skin containing these oxylipins. Here, we estimate ∼118 ± 18.1 and 104 ± 2.1 ng/mg epidermis for total 9,10-13-triHOME- and 9-HODE-EOx respectively (n = 3 ± SEM). These high levels relative to total LA-EOx detected (173 ng/mg epidermis) (quantified as per Figure 5) suggest that metabolism of LA-EOx substrate to oxygenated products is highly favoured in healthy human skin.Figure thumbnail gr5

Figure 5Analysis of triHOME, HODE and epoxy-HOME EOx ceramides and free oxylipins in healthy human epidermis. All lipids were extracted and analysed using LC/MS/MS from human epidermal breast tissue as described in Methods (n=9, mean ± S.E.M). Panel A, LA-EOx-LA ceramide lipids in healthy epidermis. Panels B-D. Epidermis levels of EOx oxidised ceramides in healthy human epidermis. Panels E-H. Free oxylipin quantification in healthy epidermis.

Free acid oxylipins were next quantified using LC/MS/MS, so that quantitative comparisons with EOx forms could be made. Only a few eicosanoids and prostaglandins were detected (Figure 5 E-H). 12-HETE (19.7 ng/mg) and 13-HODE (4.6 ng/mg) are likely originating from 12R-LOX and or 15-LOX-2 in the epidermis (Nugteren D.H. Kivits G.A. Conversion of linoleic acid and arachidonic acid by skin epidermal lipoxygenases.). Levels of 9-HODE and 13-HODE are equivalent (4.6 ng/mg) however, 9-HODE would be expected to be generated from hydrolysis from ceramides as human epidermal 12R-LOX prefers esterified linoleate as substrate (Boeglin W.E. Kim R.B. Brash A.R. A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression.). Standards used for analysis were all R enantiomers as the products were expected to be derived from the 12R-LOX pathway. However, the oxylipins quantified here could be a combination of S/R enantiomers as there would be no discernible difference in retention time during the HPLC analysis. Indeed, it has been shown that 12S-HETE is also present in epidermal tissue (Arenberger P. Kemeny L. Ruzicka T. Characterization of high-affinity 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on normal human keratinocytes.). 9,12,13-triHOME (4.4 ng/mg) and 9,10,13-triHOME (5.6 ng/mg) were detected in far lower amounts than their corresponding ceramide esters (calculated as 118 ng/mg tissue). Free 9,10-epoxy-13-hydroxy linoleate was not analysed as there is no internal standard available. The cyclooxygenase (COX) pathway products PGE2 and PGD2 were also detected at low levels, as previously reported (Leong J. Hughes-Fulford M. Rakhlin N. Habib A. Maclouf J. Goldyne M.E. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation.). Overall, this indicates that triHOMEs and HODEs predominate in skin as esterified forms in EOx. TriHOME-EOx are concentrated in the upper epidermis, while all other EOx show enrichment in deeper layers.12R-LOX is primarily found in the lowest layers of the epidermis, including the stratum basale (SB), stratum spinosum (SS) and lower stratum granulosum (SG) with the SB being lowest followed by the SS etc. eLOX3 is expressed throughout the epidermis (Li H. Lorie E.P. Fischer J. Vahlquist A. Torma H. The expression of epidermal lipoxygenases and transglutaminase-1 is perturbed by NIPAL4 mutations: indications of a common metabolic pathway essential for skin barrier homeostasis.). The location of oxidised EOx generation in skin however, is currently unknown (Epp N. Furstenberger G. Muller K. de Juanes S. Leitges M. Hausser I. et al.12R-lipoxygenase deficiency disrupts epidermal barrier function., Hansen H.S. Jensen B. von Wettstein-Knowles P. Apparent in vivo retroconversion of dietary arachidonic to linoleic acid in essential fatty acid-deficient rats., Li H. Lorie E.P. Fischer J. Vahlquist A. Torma H. The expression of epidermal lipoxygenases and transglutaminase-1 is perturbed by NIPAL4 mutations: indications of a common metabolic pathway essential for skin barrier homeostasis.). To determine this, we applied 30 tape strips to the same area of the forearm to repeatedly remove one layer of skin cells at a time. Since the uppermost layer of the skin, the stratum corneum (SC), is approximately 15 layers thick, the use of 30 strips should achieve penetration into the deeper SG and possibly the SS of the epidermis. We first determined that native LA-EOx ceramides are fairly evenly localised throughout the epidermal depth (Figure 6 A, Supplementary Figures 15A, 16A). In contrast, epoxy-HOME-ceramides (generated by 12R-LOX/eLOX3, Figure 11) increase in abundance deeper into the skin (strips 22 – 30) (Figure 6 B, Supplementary Figure 16B). Of note, epoxy-HOME EOP products were below the limit of detection. Conversely HODE-EOx and triHOME-EOx were more abundant closer to the skin surface, strips 1-18, and therefore likely to be in the upper SG and the SC (Figure 6 C-D, Supplementary Figures 15 B-C, 16 C-D). This is consistent with the location of the 12R-LOX being in the lower layers of the epidermis as the initial product is generated before rapid eLOX3 generation of the epoxy-HOME and implies that the enzyme responsible for the hydrolysis of the epoxide (recently identified as EH3) to yield the triHOME is expressed higher in the epidermis (likely the upper SG and SC)(Edin M.L. Yamanashi H. Boeglin W.E. Graves J.P. DeGraff L.M. Lih F.B. et al.Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function.). Of relevance, this coincides with keratinocyte differentiation and markers associated with CLE formation, including involucrin, transglutaminase, loricrin and filaggrin supporting a role for these ceramides in these events (Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. Transglutaminase function in epidermis.).Figure thumbnail gr6

Figure 6Human 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 ± S.E.M). Strips were combined into groups of 3 and lipids extracted and analysed using LC/MS/MS as described in Methods for total levels of lipid present. Panel A, LA-EOS is evenly distributed throughout the skin layers. Panel B. Epoxy-HOME EOS increases with greater epidermal depth and is highest in the stratum granulosum/spinosum (SG, SS) layers. Panel C,D. TriHOME EOS and HODE EOS are increased in the upper layers of epidermis and appear highest in the stratum corneum (SC).

 Human psoriatic patients have altered levels of oxidized and native ceramides both in lesions and non-lesion healthy skin.Elevated levels of free and esterified 9-HODE and 13-HODE have been previously detected in psoriatic lesions with 9R and 13S enantiomers predominating (Baer A.N. Costello P.B. Green F.A. Stereospecificity of the products of the fatty acid oxygenases derived from psoriatic scales.). To extend this to the study of specific ceramides, we determined the relative abundance of oxidised EOx identified herein. Two different comparisons were used. First, psoriatic plaques (left or right outer elbow) and uninvolved lesion-free skin (volar forearm) were tape stripped from the same patients. This allowed comparison within the same person for diseased versus non-lesion skin. Next, outer elbows (left or right) from healthy volunteers were tape stripped. These were then compared directly with psoriatic lesions that come from the same site in patients, since it is recognised that skin at different sites may show a distinct lipid profile. Data for LA, TriHOME, epoxyHOME and HODE isomers of C30, C32, C34-EOX (EOS, EOP, EOH) are shown (Figure 7, Supplementary Figures 17-20). Epoxy-HOME-EOx were below the limit of detection. LA containing C30, 32 and 34-EOx ceramides were significantly higher in psoriatic patient skin, both for the forearm (no lesion, ∼10-fold elevated) and elbow (plaque lesion, ∼5-fold elevated) (Figure 7A, Supplementary Figures 17-20). Thus, substrate ceramides are elevated in psoriatic patient skin regardless of whether a psoriasis plaque is present. Hyperproliferation of the SG and lack of terminal differentiation, providing increased abundance of EOS-producing cells in psoriasis plaques is known, providing a potential explanation (Wraight C.J. White P.J. McKean S.C. Fogarty R.D. Venables D.J. Liepe I.J. et al.Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides.). However, this was unexpected in unaffected skin in psoriasis where there is a lack of concomitant hyperproliferation. In contrast, triHOME-C32-EOS was significantly reduced in psoriatic patient skin (lesions or non-lesional skin) versus healthy controls (Figure 7 B). This contrast is also seen with triHOME-C34-EOS, triHOME-C30/C32/C34-EOH and triHOME-C34-EOP (Supplementary Figures 17-20). Last, HODE-C32-EOS were similar across forearm and elbow in healthy subjects, but for psoriatic patients they were low in unaffected forearm skin but far higher and more variable in involved elbow skin (Figure 7C). This variability is also evident with the C30/C34-EOS/EOP and EOH species with no clear significance between elbow and forearm (Supplementary Figures 18-20). There is a significant difference in HODE-C34-EOS species with this lipid being significantly higher in healthy volunteers at both sample sites (Supplementary Figure 16C). The overall pattern is that substrate EOS was significantly elevated in patient skin versus healthy controls, regardless of whether there was a lesion, while oxidized products were either significantly lower (non-lesion) or similar (lesions) in patient skin.Figure thumbnail gr7

Figure 7Lipidomics 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 analysed as described in Methods. Increasing tape strip number corresponds to increasing epidermal depth. Psoriatic patients (empty bars), healthy controls (grey bars), mean ± S.E.M.* = p<0.05, ** = p<0.01, *** = p<0.001. The bold down arrow (↓) corresponds to skin lesion samples. Geometric shapes show outliers. Panel A, epidermal tape strip profile of C30 and C32 EOS. Panel B, epidermal tape strip profile of triHOME EOS. Panel C, epidermal tape strip profile of 9-HODE EOS in psoriatic vs healthy control.

Free oxylipins were analysed in psoriatic and healthy skin, to directly compare with EOS. 9- or 13-HODE, and 9,10,13- or 9,12,13-triHOMEs were significantly increased in lesions of psoriatic patients versus healthy volunteer elbows (Figure 8 A-D). Due to unavailability of standards, we were unable to quantify epoxyHOMEs. 9-HODE and 13-HODE were significantly higher in psoriatic lesions compared to matched forearm skin matched from the same patient, as well as being significantly higher than uninvolved patient skin (Sorokin A.V. Domenichiello A.F. Dey A.K. Yuan Z.X. Goyal A. Rose S.M. et al.Bioactive Lipid Mediator Profiles in Human Psoriasis Skin and Blood.) (Figure 8 A,B). The abundant 13-HODE (which has been shown to be primarily 13S (Baer A.N. Costello P.B. Green F.A. Stereospecificity of the products of the fatty acid oxygenases derived from psoriatic scales.)) is partly generated by a 15-LOX, either 15-LOX-1 or 15-LOX-2 (Nugteren D.H. Kivits G.A. Conversion of linoleic acid and arachidonic acid by skin epidermal lipoxygenases., Baer A.N. Costello P.B. Green F.A. Free and esterified 13(R,S)-hydroxyoctadecadienoic acids: principal oxygenase products in psoriatic skin scales.). 9,10,13-triHOME and 9,12,13-triHOME showed the same trend as HODEs (Figure 8 C,D). In skin, it is well known that 12R-LOX has very poor activity towards free LA, thus, these oxidation products most likely arise following hydrolysis of the oxidized fatty acyl from the LOX-oxidized EOx (Yu Z. Schneider C. Boeglin W.E. Brash A.R. Human and mouse eLOX3 have distinct substrate specificities: implications for their linkage with lipoxygenases in skin.). In summary, free oxylipins are present at a significantly higher levels in lesional epidermis compared to both non-lesional epidermis from the same patient or compared to epidermis of healthy controls.Figure thumbnail gr8

Figure 8Free 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 from healthy controls (n = 5). From each site, 9 tape strips were obtained and pooled into groups of 3. Lipids were extracted and analysed as described in Methods. Increasing tape strip number corresponds to increasing epidermal depth. Psoriatic patients (empty bars), healthy controls (grey bars), mean ± S.E.M.* = p<0.05, ** = p<0.01, *** = p<0.001. The bold down arrow (↓) corresponds to skin lesion samples. Geometric shapes show outliers. Panel A, 9-HODE. Panel B. 13-HODE. Panel C. 9,10,13 triHOME. Panel D. 9,12,13 triHOME.

 The biosynthetic and metabolic pathway for oxidized EOx is highly upregulated in psoriatic lesionsLipidomics of psoriasis lesions demonstrated distinctive native and oxidized EOx profiles. This could result from altered expression and/or activity of the genes involved in their biosynthesis or degradation. To investigate this, a transcriptomic study on psoriatic lesions (PP) compared to healthy skin from the patient (PN) or healthy donors (NN) was downloaded and analysed for differentially expressed genes relevant to EOx metabolism (Gudjonsson J.E. Ding J. Li X. Nair R.P. Tejasvi T. Qin Z.S. et al.Global gene expression analysis reveals evidence for decreased lipid biosynthesis and increased innate immunity in uninvolved psoriatic skin.). Genes included in the analysis are labelled on Figure 12. Notably, almost all genes that regulate the biosynthesis, metabolism and coupling of EOx to form the CLE were significantly upregulated in this dataset when psoriatic lesions are compared with either uninvolved skin in the same patients, or with control skin from healthy volunteers (Figure 12, Figure 9 A, Supplementary Figure 21). This indicates that the oxidized EOx gene network that is required for forming an epidermal barrier is highly upregulated in psoriasis.Figure thumbnail gr9Figure 9Several 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 () analysed(17). RMA-normalised 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. Panel 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. Panel B. IPA analysis 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 (adj.pval < 0.01).Next, the psoriasis dataset was analysed using Ingenuity Pathway Analysis (comparing PP with NN). Here, ALOX12B was found to link with two other highly upregulated genes in the pathway that forms and couples these lipids to form the CLE, specifically TGM1 and ALOXE3, as shown (Figure 9 B). These three genes linked directly through the IPA network to ZIC1, a zinc finger protein transcription factor (Figure 9A,B) (Huttlin E.L. Bruckner R.J. Paulo J.A. Cannon J.R. Ting L. Baltier K. et al.Architecture of the human interacto

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