Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

Flaky tail (ft/ft) mice are a model of ADL that harbor two gene mutations, that are, mutations of Flg and Tmem79 (; ; ; ). The skin of ft/ft mice displays impaired permeability barrier function, as measured by increased transepidermal water loss (TEWL) (Figure 1a) (; ), which is also observed in human ADL skin regardless of disease severity, in contrast to nonlesional AD skin (; ). To validate ft/ft mice as a model of ADL with respect to lipid composition, we first studied the chain length distribution in (i) epidermal Cers containing a C₁₈ sphingosine base linked to a nonhydroxy FA (i.e., Cer[NS]), (ii) free FAs (FFAs), and (iii) very long chain, ester-linked ω-O-acylCers (i.e., Cer[EO]). We found that ft/ft mouse epidermis has a marked increase in the relative levels of Cer(NS)-containing C₁₆–C₂₂ FA moieties and a decrease in those harboring C₂₄ and C₂₆ very-long-chain fatty acid (VLCFA) moieties, whereas total Cer(NS) content remained unchanged (Figure 1b and c). Similarly, ω-O-acylCers contained diminished levels of 53:3 and 54:3 species and increased levels of several species with shorter chain lengths, without significant changes in the total amounts (Figure 1d and e). Thus, our data reveal alterations in Cer classes in the epidermis of ft/ft mice similar to those described in human ADL (; ; ; ). Moreover, the relative amounts of saturated FAs with 20–22 carbon atoms were higher and amounts of those with 26 carbon atoms were lower in the epidermis of ft/ft mice than in the epidermis of control mice (Figure 1f). Furthermore, consistent with previous observations on SC or epidermal specimens of ADL (; ), we found higher total levels of monounsaturated FAs in the epidermis of ft/ft mice than those in the epidermis of control mice, in contrast to the total levels of saturated FAs (Figure 1g). Shortening of the FA moiety in Cers has been shown to alter the formation of SC lamellar bilayers, a process initiated in LBs (; ; ). Ultrastructural analysis showed LBs to be scarce, empty, or filled with inhomogeneous material and to exhibit altered morphology in the epidermis of ft/ft mice, indicating abnormal cargo composition and altered lamellar bilayers in the SC (Figure 1h), as reported earlier (). Thus, abnormalities of epidermal lipid composition and of the LB secretory system in ft/ft mice largely mimic those observed in human ADL (; ; ; ; ; ).

To better understand the origin of the abnormal lipid composition in ADL, we examined the expression of 86 genes involved in lipid metabolism using microarray analysis. We found that genes involved in long-chain FA oxidation, activation, and trafficking were among the most strongly upregulated in the epidermis of ft/ft mice when compared with the epidermis of control mice (Figure 2a). In contrast, the expression of several genes involved in long-chain FA import into cells (Fabp3, Slc27a1, Slc27a2, Slc27a5, Cd36, Acsl3), and peroxisome proliferator–activated receptor (PPAR) signaling (e.g., Ppara, Pparg, Cpt1a, Apoc3, Apoe) was downregulated in the epidermis of ft/ft mice when compared with the epidermis of control mice (Figure 2a). qPCR confirmed increased mRNA level of Acox1 (an enzyme catalyzing the oxidation of straight-chain VLCFAs and ultra long-chain FAs [ULCFAs] in peroxisomes) and of Hsd17b4 (an enzyme participating in the second step of peroxisomal VLCFA oxidation []) in the epidermis of ft/ft mice when compared with the epidermis of the controls (Figure 2b). Moreover, ACOX1 was induced at the protein level, mainly in the upper spinous and granular layers of ft/ft mouse epidermis (Figure 2c and d). Furthermore, we measured ACOX enzymatic activity by following the production rate of hydrogen peroxide after the addition of very-long-chain fatty acyl-coenzyme As (CoAs) (). We used lignoceroyl-CoA (C_24:0-CoA) and hexacosanoyl-CoA (C_26:0-CoA), which are VLCFAs exclusively oxidized in peroxisomes (; ; ). Results revealed a significant increase in the oxidation of both fatty acyl-CoA species in epidermal bulk cells of ft/ft mice compared with that in the epidermal bluk cells of the controls (Figure 2e). Thus, peroxisomal oxidation of VLCFAs is enhanced in ft/ft mouse epidermis. In line with this observation, we found increased expression levels of Acot5 and Acot8 (Figure 2f), genes encoding two acyl-CoA thioesterases involved in downstream steps of peroxisomal FA oxidation (), as well as the expression level of Crot, encoding a carnitine octanoyltransferase responsible for the transport of shortened FAs out of peroxisomes (Figure 2g) (; ). Thus, in a context where FA uptake is not coordinated to sustained peroxisomal lipid oxidation, VLCFAs/ULCFAs might not be sufficiently packed into LBs and integrated into structural lipids such as Cers, thereby potentially explaining the shift toward the shorter acyl chain lengths of Cers and FFAs in ft/ft mouse epidermis.

PPARs are master regulators of lipid homeostasis, and reduced levels of PPARA and PPARG have been consistently observed in ADL (; ), whereas data on PPARD have not been reported. We found selective upregulation of Ppard expression in ft/ft mouse epidermis compared with that in control epidermis, as opposed to the expression of Ppara and Pparg (Figure 2a and h). In mouse KCs, Ppard expression has been shown to be upregulated by TNF-α () and IL-1β (), cytokines increased in the epidermis of ft/ft mice (Figure 3a and b), likely in response to the impaired epidermal barrier (; ). FABP5 has been shown to translocate to the nucleus to deliver ligands specifically to PPARδ and to be a PPARδ target gene (; ). FABP5 was increased at both protein and mRNA levels (Figure 3c and d) and mainly localized to suprabasal KC nuclei in the epidermis of ft/ft mice compared with those in the epidermis of the controls (Figure 3e). Thus, enhanced PPARδ signaling might promote peroxisomal oxidation of VLCFAs/ULCFAs in ft/ft mouse epidermis by the upregulation of ACOX1 (; ).

To study the relevance of our findings in humans, we carried out immunostaining for ACOX1 in the skin of patients with AD and in those of healthy donors. We found a marked increase of ACOX1 in the spinous and granular layers of ADL epidermis compared with those in the healthy skin (Figure 4a), in contrast to nonlesional AD skin (Figure 4b). To investigate how increased ACOX1 in the human epidermis might contribute to ADL, we overexpressed ACOX1 in human KCs. Cells were infected with lentivirus particles encoding cDNA for human ACOX1 (pHR-SFFV-ACOX1) or puromycine resistance (pHR-SFFV-Puro [control]) before generating human epidermal equivalents (HEEs) (Figure 4c). Overexpression of ACOX1 was demonstrated by qPCR, Western blot analysis, and immunofluorescence staining (Figure 4d–f). Transmission electron microscopy revealed an overall decrease in the number of LBs in HEEs overexpressing ACOX1 compared with that in the Puro controls (Figure 5a and b). Moreover, ACOX1 overexpression was associated with reduced LB secretion and altered LB content, with many LBs containing inhomogeneous material or appearing as empty vesicles, in contrast to the Puro controls (Figure 5a–c). However, transepithelial electrical resistance did not differ, and Lucifer yellow did not penetrate the living layers of HEEs in both experimental groups (Figure 5d and e). This is in line with previous work showing no Lucifer yellow penetration in AD HEEs despite ultrastructural abnormalities of the SC and of the LB secretion system (; ). Furthermore, Ki67 staining showed that ACOX1 overexpression resulted in enhanced KC proliferation, but no noticeable histological differences were observed (Figure 5f). We screened the expression of various inflammatory mediators (i.e., IL1A, IL1B, IL8, IL18, IL33, TSLP, TNFA, S100A8, S100A9) and found a 1.9-fold increase in IL8 mRNA level and a 1.8-fold increase in TNFA mRNA level, although these differences did not reach significance (Figure 5g). We did not observe differences in the expression of inflammatory cytokines between nontransfected and Puro control HEEs (data not shown). Furthermore, lipidomic analysis showed that relative levels of Cer(NS) containing C_24:0 and C_26:0 FA moiety were increased, whereas those containing C_24:1 (nervonic acid) and C_26:1 (hexacosenoic acid) were reduced in HEEs overexpressing ACOX1 compared with those in the Puro controls (Figure 6a), similar to FFA profile (Figure 6b). Nervonic and hexacosenoic acids are exclusively oxidized in peroxisomes and contribute to epidermal barrier formation (). Overall, these results show that ACOX1 overexpression enhances KC proliferation, potentially through IL8 and TNFA upregulation (; ), and concomitantly inhibits the LB secretion system.

Recent research suggested that altered lipid biosynthesis as well as hampered FA elongation upstream of Cer synthesis may underlie the abnormal lipid profile of AD skin (). Therefore, we measured the mRNA levels of genes encoding enzymes involved in FA synthesis and elongation and found that expressions of Fasn, Elovl1, and Elovl4 were increased and that of Elovl6 was decreased in the epidermis of ft/ft mice compared with those in the epidermis of the controls (Figure 7a). Accordingly, the protein level of ELOVL1 was increased in ft/ft mouse epidermis (Figure 7b). Thus, these results show that FA synthesis and elongation are triggered in ft/ft mouse epidermis. Because ELOVL1 and ELOVL4 elongate FAs from C₁₈ up to C₃₈, which are preferential substrates for peroxisomal oxidation (; ; ), it is likely that VLCFA synthesis and peroxisomal β-oxidation are coupled in ft/ft mouse epidermis. Because epidermal FFA composition exhibits a shift toward shorter FAs (Figure 1f), enhanced VLCFA synthesis in ft/ft mouse epidermis might not be able to compensate for their forced peroxisomal oxidation. Increased TEWL (Figure 1a) provokes heat loss (; ; ), and interestingly, peroxisomal β-oxidation of FAs per se generates heat and is involved in adaptive thermogenesis independent of UCP1 (; ). To investigate the relationship between TEWL and ACOX1, we applied an occlusive dressing onto ft/ft mouse skin to reduce TEWL (; ). A reduction of ACOX1 at protein and mRNA levels (borderline nonsignificant) was observed in ft/ft mouse epidermis after skin occlusion (Figure 7c). Thus, in ft/ft mouse epidermis, VLCFA/ULCFA synthesis might be enhanced to provide peroxisomes with lipid substrates to fulfill bioenergetic requirements (e.g., KC proliferation/differentiation) and local thermogenesis owing to increased TEWL.

We next questioned whether glucose might serve as an energy source to sustain KC proliferation and contribute to hyperplasia in ADL epidermis, similar to what has been evidenced in psoriasis (). We assessed the expression of glucose transporters in mouse epidermis and found that mRNA levels of Glut1 and Glut3 were significantly increased in ft/ft mouse epidermis compared with those in the control epidermis, although Glut1 was the most abundant isoform (Figure 8a). Moreover, immunofluorescence staining showed a marked increase of GLUT1 in the basal layer and immediate spinous layer (; ) of ft/ft mouse epidermis compared with those of the control epidermis (Figure 8a), similar to that observed in those of the human ADL epidermis (Figure 8b). Moreover, the mRNA and protein levels of key glycolytic enzymes (HK1/2, GPI1, and, PKM) (Figure 8c and d) () as well as glucose and pyruvate uptake (Figure 8e) and adenosine triphosphate (ATP) content (Figure 8f) were all significantly increased in ft/ft mouse epidermis compared with those in the control epidermis. mRNA levels of key proteins of the pentose phosphate pathway (G6pdx, Pgd, and Tkt)—a metabolic pathway parallel to glycolysis ()—were increased in the epidermis of ft/ft mice compared with those in the epidermis of the controls (Figure 8g). Mammalian cells generate ATP from glucose through glycolysis in the cytosol and oxidative metabolism through oxidative phosphorylation in the mitochondria. We found that lactate production (Figure 9a) and the mRNA level of Ldha were higher in the epidermis of ft/ft mice than in that of the controls (Figure 9b), suggesting a higher conversion of pyruvate into lactate. Yet, metabolomic profiling revealed increased intracellular concentrations of hexose but not of lactate or pyruvate in the epidermis of ft/ft mice (Figure 9c), suggesting a rapid excretion of lactate. The expression of mitochondrial pyruvate carrier subunits Mpc1 and Mpc2 remained unchanged (Figure 9d), suggesting no increased entry of pyruvate in the mitochondria in the epidermis of ft/ft mice. Moreover, the expression of pyruvate dehydrogenase kinase isoform 1, a key enzyme involved in metabolic switch toward predominant anaerobic glycolysis through the reduction of mitochondrial oxidative pathways (Warburg effect) (), was increased in the epidermis of ft/ft mice in comparison with that of the controls (Figure 9e). In line with this, the concentrations of intermediates of the Krebs cycle were overall similar in the two groups (Figure 9c)—malic acid was increased. The Krebs cycle provides complexes I and II of the respiratory chain with reduced substrates, that is, NADH₂ and FADH₂, respectively. In line with unaltered Krebs cycle turnover, the activity of mitochondrial complexes I and II remained unchanged in the epidermis of ft/ft mice compared with that in the epidermis of the controls (Figure 9f). Our earlier results showed upregulated peroxisomal FA oxidation coupled with increased export of shortened FAs out of peroxisomes (Figure 2b–g) and increased expression of Acadm, Acadl, Acadvl, and Acad9 (Figures 2a and 9g and h), altogether suggesting possible enhancement of mitochondrial oxidation of FAs (; ). However, the enzymatic activity of mitochondrial ACADL was reduced in ft/ft mouse epidermis when compared with control epidermis (Figure 9i) as well as the expression of Cpt1a (Figures 2a and 9j). Thus, in ft/ft mouse epidermis, shortened FAs secreted out of the peroxisomes might be partly oxidized in mitochondria and might partly be used for synthesizing shorter Cers. Because these results show increased anaerobic glycolysis in ft/ft mouse epidermis, we next investigated the dependency of AD-like phenotype of ft/ft mice on this metabolic pathway. Ears of ft/ft mice were topically treated with WZB-117, a molecule blocking glucose uptake and glycolysis through GLUT1 inhibition (; ). Treatment with WZB-117 reduced mRNA and protein levels of GLUT1 (Figure 10a and b) and decreased mRNA levels of the key glycolytic genes Hk2, Gpi1, Pkm, and Ldha in the epidermis of ft/ft mice (Figure 10c and d). Moreover, topical treatment of ft/ft mice with WZB-177 significantly reduced epidermal hyperplasia as well as the mRNA level of Keratin 16, that is, K16) (Figure 10e and f) and the number of Ki67⁺ KCs (Figure 10g). Treatment of ft/ft mice with WZB-177 led to a drop in epidermal intracellular ATP content (Figure 10h) but did not reduce the epidermal expression of markers of barrier impairment such as Il1a, Il1b, and Tnfa or the expression of the T helper type-2 cytokine Il13 (Figure 10i and j). Collectively, these results suggest that in ft/ft mouse epidermis, glycolysis and the pentose phosphate pathways—likely not mitochondria—provide KCs with ATP and macromolecules to fulfill high bioenergetic needs to sustain a high rate of proliferation. However, enhanced glycolysis does not contribute to local inflammation.

Because ft/ft mice carry two gene mutations, that is, Flg and Tmem79 (), we next sought to determine the contribution of FLG deficiency to abnormalities in lipid metabolism using Flg-knockout (KO) mice (; ). Total amounts of Cer(NS) and monounsaturated FAs were increased in the epidermis of Flg-KO mice when compared with that of the controls in absence of changes in chain length distribution of epidermal lipids (Figure 11a–c). Moreover, the mRNA levels of all Ppar isoforms as well as the mRNA level, protein abundance, and activity of ACOX1 were unaltered in the Flg-KO mouse epidermis compared with those in the control epidermis (Figure 12a–d). The expression of most lipid metabolic genes was reduced, with the exception of Fabp expression, which was moderately increased (Figure 12a). Despite normal lipid composition, transmission electron microscopy showed a heterogeneous population of LBs exhibiting both normal and abnormal content and secretion in Flg-KO mouse epidermis, albeit less pronounced than in the epidermis of ft/ft mice (Figure 12e). Furthermore, the mRNA level of Glut1 was unaltered, and no changes in glucose uptake or lactate production were observed in the Flg-KO mouse epidermis compared with those in the control epidermis (Figure 12f and g). Metabolomic profiling of the epidermis revealed low intracellular concentrations of pyruvate but unchanged levels of hexose and lactate in Flg-KO mice compared with those in the controls (Figure 12h). Moreover, concentrations of the intermediates of the Krebs cycle were reduced or unchanged in the Flg-KO epidermis compared with those in the control epidermis (Figure 12h). Thus, FLG deficiency per se does not alter lipid or glucose metabolism in KCs, and the abnormalities in LB cargo composition observed in FLG-deficient mice, similar to what has been observed in patients with various ichthyoses, might rather result from cytoskeletal abnormalities, as suggested earlier (; ; ). Moreover, our results are in line with those of previous work showing that the lipid phenotype of atopic skin is independent of FLG status (; ).

To investigate whether the observed metabolic changes were specific to ft/ft mice, we utilized another mouse model of ADL exhibiting increased TEWL, that is, mice topically treated with MC903 (Figure 13a and b) (; ; ). We found increased ACOX1 levels in the granular KCs of the epidermis of MC903-treated mice compared with those in the epidermis of vehicle-treated controls, despite an unchanged mRNA level (Figure 13c). Furthermore, MC903 treatment enhanced epidermal expression of Acot5, Crot (Figure 13d and e), Ppard, and Fabp5, as opposed to the expression of Ppara or Pparg (Figure 13f). With respect to glycolysis, MC903-treated mice displayed increased epidermal GLUT1 (Figure 13g) and expression of key glycolysis-related enzymes (Hk2, Gpi1 and Pkm, Ldha, Pdk1) (Figure 13h and i). Thus, these results suggest that enhanced peroxisomal β-oxidation and anaerobic glycolysis are not specific to ft/ft mice and are independent of Tmem79 mutation.

Because ultra-long Cers such as C₂₆ Cers are decreased in the epidermis of patients with psoriasis (; ; ), we wondered whether ACOX1 could be enhanced in psoriatic epidermis. Mice topically treated with imiquimod (IMQ) exhibited psoriasiform inflammation associated with increased TEWL (Figure 14a and b) as reported earlier (; ; ). Despite unchanged mRNA level (Figure 14c), immunostaining revealed enhanced ACOX1 in the upper epidermis of IMQ-treated mice compared with that in the upper epidermis of petroleum jelly-treated controls (Figure 14c), similar to that observed in human lesional psoriatic epidemis (Figure 14d). These results might confirm a tight relationship between TEWL and ACOX1.

Ft/ft mice were bred on a pure C57BL/6 background (kind gift from M. Amagai). Mice of the inbred strain C57BL/6 were purchased from Charles River Laboratories (Sulzfeld, Germany). Flg-KO mice (BRC number 05850: B6.Cg-Flg<tm1>) on a pure C57BL/6 background were purchased from RIKEN (Tsukuba, Japan). Age-matched female mice were used at age >24 weeks. Mouse care and use were approved by the Austrian Federal Ministry of Science and Research and were performed according to institutional guidelines (BMWFW-66.011/0199-WF/V/3b/2016).

Mouse epidermis was isolated by trypsinization (Sigma-Aldrich, St. Louis, MO) for 30 minutes at room temperature. For experiments using dorsal skin, mice were shaved, and their hair was removed using Veet hair removal cream 72 hours before sample collection. KCs were isolated and cultured as described earlier ().

For inhibition of glucose transport, 1 mg/ml WZB-117 dissolved in acetone (Sigma-Aldrich) or vehicle (100% acetone) was applied topically once daily onto the ears of female ft/ft mice for 9 days according to previous work (). The epidermis was collected 6 hours after the last treatment. Psoriasis-like skin inflammation was induced in adult, age-matched, female C57BL/6 mice by topical application of a daily dose of 12.5 mg of Aldara cream (Meda Pharma GmbH, Frankfurt, Germany), containing 5% IMQ onto ears for 5 days (). Control mice were topically treated with petroleum jelly. The epidermis was collected 24 hours after the last treatment. To induce ADL, 45 μM MC903 (Sigma Aldrich) in 12.5 μl ethanol was topically applied onto both sides of the ears of female C57BL/6 mice daily for 9 days consecutively (). Control mice were topically treated with ethanol (vehicle). The epidermis was collected 24 hours after the last treatment.

Hair was removed from the dorsal skin of mice (shaving followed by depilation), and a latex membrane was applied on the upper half part of the back as described previously (; ). The lower half of the back was left uncovered and was used for each mouse as its own control. After 4 days, mice were killed, and the skin was harvested.

Skin biopsies were taken from nonlesional or lesional (chronic lesions) skin of adult European patients with AD or from lesional skin of adult European patients with psoriasis. Abdominal or breast skin obtained from plastic surgery procedures of adult European subjects was used as healthy control tissue. Human subjects gave skin samples voluntarily after written informed consent. Alternatively, paraffin blocks were used in the frame of a retrospective study. The study was approved by the Ethics Committee of the Medical University of Innsbruck (Innsbruck, Austria) and was conducted in accordance with the Declaration of Helsinki principles (UN4253-297/4.18, UN5073-325/4.2, AN2016-0260-368/4.22-396/5.14 [4117a], EK1412/2220). The number and characteristics of patients are summarized in Table 1.

The epidermis of mouse ears was stored overnight in RNAlater Stabilization Solution (Invitrogen, Carlsbad, CA) at 4 °C. Total RNA was then extracted, and cDNA was prepared according to a previous protocol (). Primers were purchased from Applied Biosystems (Foster City, CA) (Table 2). TBP and PPIA were used for normalization. Expression fold changes were calculated using the 2^–ΔΔCt formula. For pathway-focused gene expression analysis, equal amounts of RNA from each mouse within groups were pooled, and samples were prepared for the PPAR Targets RT² Profiler PCR Array (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR array results for selected genes were validated by qPCR analysis from individual samples. Data are expressed as a heat map, where fold changes of the expression level of genes in a pool of three mice are shown and normalized to the values of control mice. The data shown represent a subset of the entire 89 genes featured in the array, selected for genes involved in FA metabolism. The full gene list is provided in Table 3.

Mouse ears and HEEs were fixed in 4% formaldehyde and embedded in paraffin. Sections of 3 μm thickness were obtained and stained with H&E for routine histology. Immunostaining with anti‒Ki-67 antibody (Roche, Basel, Switzerland) was used to evaluate KC proliferation. Analysis was performed using an Olympus BH-2 light microscope (Olympus, Tokyo, Japan) equipped with a ProgRes C10plus camera and ProgRes CapturePro 2.8.8 image analysis software (Jenoptik, Jena, Germany). For immunofluorescence, sections (3 μm) were deparaffinized, and antigen retrieval was facilitated using 10 mM citrate buffer containing 0.5% Tween (pH 6.0). Sections were blocked with 10% goat serum and thereafter incubated with primary antibodies in 2% goat serum, washed, and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (Invitrogen) in 2% BSA. Nuclei were counterstained with DAPI (Invitrogen). Images were acquired with a spinning disk confocal system (UltraVIEW VoX; Perkin Elmer, Waltham, MA) connected to a Zeiss Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany). To determine nuclear localization, nuclei were counterstained with DAPI (Invitrogen), and images were analyzed using GNU Octave software. Channels were merged, and mean fluorescence intensities of the green stain were assigned to gray levels in a gray scale in areas where the green channel exhibited overlap with DAPI nuclear stain. Mean intensities are represented by the spectrum of pseudocolors, ascending from black to white.

For western blot analysis, epidermal sheets or HEEs were lysed in modified RIPA lysis buffer (10 mM Tris-hydrochloride, 150 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4) in the presence of protease and phosphatase inhibitors (cOmplete Mini and PhosSTOP; Roche). Membranes were incubated overnight with primary antibodies, and bands were detected with Alexa Fluor680 goat anti-rabbit (Invitrogen) or IRDye 800CW goat anti-mouse (Li-COR Biosciences, Lincoln, NE) secondary antibodies. Blots were scanned with an LI-COR Biosciences analyzer. β-Actin was used as a loading control.

The following primary antibodies were used for western blots and immunofluorescence staining: anti-ACOX1 (ab184032) and anti‒β-actin (ab8227) from Abcam (Cambridge, United Kingdom), anti-ELOVL1 (PA5-39159), anti-GLUT1 (RB-9052-P0) from Thermo Fisher Scientific (Waltham, MA), anti-HK1 (#2024) and pyruvate kinase 1/2 (#3190) from Cell Signaling Technology (Danvers, MA), and anti-FABP5 (12348-1-AP) from Proteintech Group (Rosemont, IL).

The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1. Sequence-verified lentiviral plasmid was used to generate lentiviral particles. The promoter is a spleen focus forming virus strain P long terminal repeat sequence (SFFV-U3LTR). The original vector has been described in the study by .

Lentiviral transduction of target cells was performed as described previously (). In brief, confluent HEK293T cells were transfected with a 1.5 μg lentiviral vector (pHR-SFFV-hACOX1 or pHR-SFFV-Puro) and 0.9 μg of each packaging plasmid (pSPAX2, pMD-G VSV-G, both vectors were kindly provided by D. Trono [École Polytechnique fédérale de Lausanne, Switzerland]) by calcium phosphate‒based transfection. Virus-containing cell supernatant was harvested after 48 hours, filtered through a 0.2 μm filter, diluted at 1:2 with fresh CnT-basal KC medium (CnT-BM.1) supplemented with human KC growth supplements (CnT-07) (CELLnTEC Advanced Cell Systems, Bern, Switzerland), and added to passage 2 KCs in the presence of 1 μg/ml polybrene.

To generate HEEs, primary human KCs were trypsinized 72 hours after infection and seeded at a density of 3.4 × 10⁵ on inserts with a pore size of 0.4 μm (Merck Millipore, Burlington, MA) in CnT-basal KC medium (CnT-BM.1) supplemented with human KC growth supplements (CnT-07) (CELLnTEC Advanced Cell Systems) (). After 48 hours, the medium was switched to a mixture of CnT-PR-3D medium (CELLnTEC Advanced Cell Systems) and DMEM (Lonza, Basel, Switzerland) medium (60:40 [v/v]) according to the procedure by . After 16 hours, HEEs were lifted to the air‒liquid interface and cultivated at a humidity of 55–60% at 37 °C and 5% carbon dioxide for an additional 8 days until harvesting. All cell cultures were grown in the absence of antibiotics and antifungals.

Punch biopsies of 3 mm diameter were taken from mouse ears or from HEEs and fixed in Karnovsky’s buffer for 1 hour at room temperature, stored overnight at 4 °C, and then rinsed twice in 0.1 M cacodylate buffer. Thereafter, samples were processed using osmium tetroxide postfixation staining and visualized with a Zeiss 10A (Carl Zeiss, Jena, Germany) or a Hitachi HT 7700 (Tokyo, Japan) electron microscope as described previously ().

LB density was defined as the number of LBs counted per unit area (μm²) of the cytoplasm of granular KCs. To quantify secretion areas, the area of secreted content (in nm²) was measured over the length of the stratum granulosum‒SC interface (in μm). All image quantifications were performed using ImageJ software.

Transepithelial electrical resistance was measured on HEEs on day 10 with an EVOM3 device (World Precision Instruments, Sarasota, FL) according to the manufacturer’s instruction. Briefly, measurements were performed using 500 μl of a mixture of fresh CnT-PR-3D medium (CELLnTEC Advanced Cell Systems) and DMEM (Lonza) medium (60:40 [v/v]) on top of the inserts and 1,500 μl below the inserts. An insert not seeded with cells was used as a blank. Results are expressed as Ohm/cm².

Penetration of Lucifer yellow was performed as described previously (). Briefly, 200 μl of 1 mM Lucifer yellow (Sigma-Aldrich) was applied onto HEEs and incubated for 2 hours at 37 °C. Then, HEEs were rinsed with PBS, fixed in formaldehyde, and embedded in paraffin. Thereafter, 6 μm sections were cut, deparaffinized, counterstained with DAPI, and analyzed with an Olympus BX60 epifluorescence microscope (Olympus).

TEWL was measured with a device from Courage + Khasaka (MP6, Cologne, Germany). Measurements were carried out under light ketamine anesthesia and repeated three times on each ear. The mean values of both ears were used for analysis. Temperature and relative humidity were controlled during the experiment.

Epidermal thickness was evaluated on H&E-stained tissue sections using ProgRes CapturePro 2.8.8 image analysis software (Jenoptik). For each mouse, serial measurements were performed at 100 μm intervals on three different areas (from the stratum basale to the stratum granulosum).

Epidermal lipids were extracted from snap-frozen epidermis obtained by trypsinization from the dorsal skin of mice. Samples were weighed and extracted according to the procedure used by in 700 μl methyl-tert-butyl ether/methanol (3/1, v/v) containing 500 pmol butylated hydroxytoluene, 1% acetic acid, and internal standards (1.4 nmol of FA 17:0 and FA 21:0; 400 pmol of d18:1/17:0 Cer, Avanti Polar Lipids, Alabaster, AL). Total lipid extraction was performed under constant shaking for 30 minutes at room temperature. After the addition of 140 μl H₂O and further incubation for 30 minutes at room temperature, samples were centrifuged at 1,000g for 10 minutes. A total of 500 μl of the upper, organic phase was collected and dried under a stream of nitrogen. Lipids were resolved in 500 μl 2-propanol/methanol/H₂O (7/2.5/1, v/v/v) for ultra-HPLC-quadrupole time-of-flight mass spectrometry analysis. A total of 50 μl of resolved lipids were used for the derivatization of FAs using the AMP + MaxSpec Kit (Cayman Chemical, Ann Abor, MI) according to the manufacturer’s instructions. The remaining epidermal sheets were solubilized in 0.3N sodium hydroxide at 65 °C for 4 hours, and the protein content was determined using Pierce BCA reagent (Thermo Fisher Scientific) and BSA as a standard.

Chromatographic separation was performed using an AQUITY-UPLC system (Waters Milford, MA) as described () with two modifications: (i) a Luna ω-C18 column (2.1 × 50 mm, 1.6 μm; Phenomenex, Torrance, CA) was employed and (ii) a 20-minute linear gradient was started with 80% solvent A (methanol/H₂O, 1/1, v/v; 10 mM ammonium acetate, 0.1% formic acid, 8 μM phosphoric acid). A SYNAPTG1 qTOF HD mass spectrometer (Waters) equipped with an electrospray ionization source was used for the detection of lipids in positive ionization mode. Data were acquired using MassLynx 4.1 Software (Waters). Lipids were manually identified and analyzed with Lipid Data Analyzer 2.5.3 software (). Data were normalized for recovery, extraction, and ionization efficacy by calculating analyte–to–internal standard ratios (arbitrary unit) and expressed as AU/mg protein.

ATP content in epidermal cell pellets was quantified using the ATPlite 1step (Perkin Elmer) assay kit. Luminescence was read with a Tecan Infinite Pro 200 reader. Values were normalized to total protein content.

Punch biopsies taken from the dorsal skin of the mice were trypsinized, and the obtained epidermal sheets were incubated in 250 μl CnT-Prime 3D Barrier culture medium (CELLnTEC Advanced Cell Systems) in duplicate at 37 °C and 5% carbon dioxide. After 24 hours, the medium was collected, deproteinized using a 10 kDa MWCO spin filter, and assayed for glucose and pyruvate concentration using a colorimetric assay kit (Sigma-Aldrich). The concentration of lactate in the medium was evaluated as previously described (). Fresh medium was used in all the assays as a reference. Results were normalized by the total protein content of the tissue.

Epidermal cell suspensions from mouse ears were prepared as reported earlier (). Cells were counted, pelleted, and frozen in liquid nitrogen. On the day of analysis, cell pellets were resuspended in Buffer A (250 mM sucrose, 20 mM Tris-hydrochloride, 2 mM EDTA, 1 mg/ml BSA, pH 7.2). To promote membrane permeabilization, samples were subjected to one cycle of snap freezing/thawing. Thereafter, mitochondria and peroxisomes were sedimented by centrifugation at 13,000g for 1.5 minutes at 4 °C. Pellets enriched in mitochondria and peroxisomes were resuspended in Buffer A. The efficacy of organelle enrichment was controlled by western blot analysis. The activity of acyl-CoA oxidase was measured by a fluorometric-based method with some modifications (). Briefly, the assay medium contained 50 mM Tris-hydrochloride buffer (pH 8.0), 1 U/ml horseradish peroxidase type II (Sigma-Aldrich), 2 μM BSA, 3 μM flavin-adenin-dinukleotid (Sigma-Aldrich), and 40 mM aminotriazole (Sigma-Aldrich) to inhibit endogenous catalase. To monitor hydrogen peroxide generation, homovanillic acid was replaced by 5 μM Amplex Red (Thermo Fisher Scientific), which is more sensitive and reacts with hydrogen peroxide with a 1:1 stoichiometry to produce resorufin. About 15–20 μg of protein were preincubated with assay medium for 3 minutes at 37 °C. The reaction was initiated by the addition of 500 μM CoA esters (lignoceroyl- and hexacosanoyl-CoA from Avanti Polar Lipids), and the increase in fluorescence was recorded for 20 minutes at 37 °C, ex = 563 nm and em = 595 nm. Results were normalized by the protein content of the cell homogenates.

Complex I activity was measured using a dichlorophenolindophenol-coupled method (). In brief, cellular fractions were preincubated with assay medium containing 80 mM potassium phosphate (pH 7.4), 1 mM potassium cyanide, 2 mM sodium azide, 0.075 mM dichlorophenolindophenol, and 0.1 mM decylubiquinone. Thereafter, 0.3 mM NADH was added, and the decrease in absorbance was recorded at 600 nm. Complex II activity was measured according to the methods by ). In brief, assay medium contained 100 mM potassium phosphate (pH 7.5), 0.5 M succinate, 10 mM potassium cyanide, 20 mM phenazine, and 500 μM dichlorophenolindophenol. Succinate-dependent reduction of dichlorophenolindophenol was measured at 600 nm.

Dehydrogenation of palmitoyl-CoA mediated by mitochondrial dehydrogenases was measured according to the methods by . Briefly, cellular fractions were preincubated in 100 mM potassium phosphate buffer (pH 7.2) containing 0.2% Triton X-100, 0.1 mM EDTA, and 150 μm ferricenium ion. The reaction was initiated by the addition of 200 μM palmitoyl-CoA, and the decrease in absorbance at 300 nm was recorded for 5 minutes.

Tissue samples were homogenized using a Precellys 24 tissue homogenizer (Precellys CK14 lysing kit, Bertin Technologies, Rockville, MD). A total of 9 μl of methanol was added per mg tissue. A total of 30 μl of the homogenized tissue samples was transferred into a glass vial, and 20 μl of metabolite internal standard mix and 270 μl methanol were added. After vortexing, the mixture was incubated in a shaker for 20 minutes on ice. The samples were centrifuged at 500g for 10 minutes. The supernatant was collected and dried using a nitrogen evaporator. Afterward, samples were reconstituted in 100 μl water and were used for the metabolite analysis. A 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, CA) coupled with a 6470 triple quadrupole mass spectrometer (Agilent Technologies) was used for the liquid chromatography with tandem mass spectrometry analysis. The chromatographic separation for samples was carried out on a ZORBAX RRHD Extend-C18, 2.1 × 150 mm, 1.8 μm analytical column (Agilent Technologies). The column was maintained at a temperature of 40 °C, and 4 μl of the sample was injected per run. The mobile phase A was 3% methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid in water, and the mobile phase B was 10 mM tributylamine and 15 mM acetic acid in methanol. The gradient elution with a flow rate of 0.25 ml/minute was performed for a total time of 24 minutes. Afterward, a backflushing of the column using a 6port/2-position divert valve was carried out for 8 minutes using acetonitrile, followed by 8 minutes of column equilibration with 100% mobile phase A. The triple quadrupole mass spectrometer was operated in an electrospray ionization negative mode, spray voltage of 2 kV, gas temperature of 150 °C, gas flow of 1.3 l/min, nebulizer 45 psi, sheath gas temperature of 325 °C, and sheath gas flow of 12 l/min. The metabolites of interest were detected using a dynamic MRM mode. The MassHunter 10.0 software (Agilent Technologies) was used for the data processing. Ten-point linear calibration curves with internal standardization were constructed for the quantification of metabolites. Metabolite concentrations were expressed as pmol/mg tissue. The full list of the quantified metabolites and respective fold changes are provided in Table 4.