Lipid abnormalities in atopic skin are driven by type 2 cytokines

Abstract

Lipids in the stratum corneum of atopic dermatitis (AD) patients differ substantially in composition from healthy subjects. We hypothesized that hyperactivated type 2 immune response alters AD skin lipid metabolism. We have analyzed stratum corneum lipids from nonlesional and lesional skin of AD subjects and IL-13 skin-specific Tg mice. We also directly examined the effects of IL-4/IL-13 on human keratinocytes in vitro. Mass spectrometric analysis of lesional stratum corneum from AD subjects and IL-13 Tg mice revealed an increased proportion of short-chain (N-14:0 to N-24:0) NS ceramides, sphingomyelins, and 14:0-22:0 lysophosphatidylcholines (14:0-22:0 LPC) with a simultaneous decline in the proportion of corresponding long-chain species (N-26:0 to N-32:0 sphingolipids and 24:0-30:0 LPC) when compared with healthy controls. An increase in short-chain LPC species was also observed in nonlesional AD skin. Similar changes were observed in IL-4/IL-13-driven responses in Ca2+-differentiated human keratinocytes in vitro, all being blocked by STAT6 silencing with siRNA. RNA sequencing analysis performed on stratum corneum of AD as compared with healthy subjects identified decreased expression of fatty acid elongases ELOVL3 and ELOVL6 that contributed to observed changes in atopic skin lipids. IL-4/IL-13 also inhibited ELOVL3 and ELOVL6 expression in keratinocyte cultures in a STAT6-dependent manner. Downregulation of ELOVL3/ELOVL6 expression in keratinocytes by siRNA decreased the proportion of long-chain fatty acids globally and in sphingolipids. Thus, our data strongly support the pathogenic role of type 2 immune activation in AD skin lipid metabolism.

Keywords: Dermatology; Skin.

Figure 1. The effect of skin atopy on stratum corneum lipids.

Changes in relative level of short- and long-chain molecular species in ceramides (A), sphingomyelins (B) and lysophosphatidylcholines (C) in stratum corneum of atopic dermatitis (AD) patients as compared with skin of normal control subjects. Each lipid molecular species was quantified by targeted liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and normalized by sample total protein content, and data were expressed as relative percentage within each lipid subclass. Data are presented as box-and-whisker plot, with whiskers showing minimum and maximum values. *,⁺P < 0.05, **,⁺⁺P < 0.01, ***,⁺⁺⁺P < 0.001 as compared with normal control subjects using 2-tailed Student’s t-test. AD lesional skin (n = 15), AD nonlesional skin (n = 30), normal control skin (n = 25).

Figure 2. Volcano plot representation of lipid changes associated with atopic dermatitis in stratum corneum.

Volcano plots of changes in relative percentage in molecular species of lipids from selected individual lipid subclasses in nonlesional (A) and lesional (B) stratum corneum of atopic dermatitis (AD) patients versus normal control (CTR) subjects. Each lipid molecular species was quantified by targeted or semitargeted liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), normalized by sample total protein content, and data were expressed as relative percentage within each lipid subclass. Note that lysophosphatidylcholine (LPC) molecular species demonstrate the most striking reciprocal shift in the long-chain and short-chain molecules in lesional AD skin. AD lesional skin (n = 15), AD nonlesional skin (n = 30), normal control skin (n = 25).

Figure 3. Expression of elongation of long-chain fatty acids family member 3 and 6 (ELOVL3 and ELOVL6) enzymes is decreased in atopic skin.

Expression pattern for elongation of long-chain fatty acids family member 1–7 (ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7) (A–G) in human stratum corneum as detected by RNA-seq analysis of skin tape strip RNA samples collected from atopic dermatitis (AD) patients and normal controls (NC). Note that the expression of ELOVL3 and ELOVL6 is decreased in lesional AD skin (both elongases take part in the formation of long-chain fatty acids). AD lesional skin (n = 11), AD nonlesional skin (n = 18), NC skin (n = 13). One-way ANOVA was used for statistical analysis.

Figure 4. IL-13 driven lesion development in mouse skin profoundly affects stratum corneum lipids.

Changes in relative level of short- and long-chain molecular species in NS ceramides with C18 sphingosine (A), sphingomyelins (B), and lysophosphatidylcholines (C) in stratum corneum from nonlesional and lesional skin of IL-13 Tg mice and their littermate controls. IL-13 Tg mice spontaneously develop skin lesions. Tape strips were collected from both lesional and nonlesional areas of mouse skin. Each lipid molecular species was quantified by targeted liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and normalized by sample total protein content, and data were expressed as relative percentage within each lipid subclass. Data are presented as box-and-whisker plot, with whiskers showing minimum and maximum values. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT littermate control as determined using 2-tailed Student’s t test. IL-13 Tg lesional skin samples n = 6, IL-13 Tg nonlesional skin samples n = 8, WT control mice n = 10.

Figure 5. IL-13 downregulates skin expression of elongation of long-chain fatty acids family member 3 and 6 (ELOVL3 and ELOVL6) enzymes in mice.

Expression of IL-13 (A), elongation of long-chain fatty acids family member 3 (ELOVL3) (B), and elongation of long-chain fatty acids family member 6 (ELOVL6) (C) in skin of IL-13 Tg mice and control animals as detected by real-time PCR. An increased expression of IL-13 mRNA and significant downregulation of ELOVL3 and ELOVL6 mRNA expression was observed in lesional skin of IL-13 Tg mice. IL-13 Tg lesional skin samples (n = 8), IL-13 Tg nonlesional skin samples (n = 10), WT control mice (n = 12).

Figure 6. The effect of differentiation and type 2 cytokines on keratinocyte lipids.

(A) The effect of Ca²⁺-induced differentiation in vitro on relative proportion of selected ceramides in keratinocytes. Each ceramide molecular species was quantified by targeted liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and normalized by sample total lipid phosphorus content, and data were expressed as relative percentage within each lipid subclass. Each individual data point was expressed relative to an average of nondifferentiated control. Note the decrease in relative content of short-chain ceramides and the increase in long-chain ceramides. STAT6 controls the effect of IL-4/IL-13 on NS ceramides with C18 sphingosine (B) and sphingomyelins (C) in differentiated keratinocytes. No differences in lysophosphatidylcholines (LPC) are observed in the keratinocyte model (D). Keratinocytes were differentiated in the absence or presence of IL-4/IL-13. Keratinocytes were treated with STAT6 siRNA during a 5-day differentiation period. Lipids were quantified by LC-ESI-MS/MS and normalized by total lipid phosphorus content (n = 3). One from 2 typical experiments is shown; each treatment condition done in triplicates. Two-tailed Student’s t test was used for statistical analysis.

Figure 7. Type 2 cytokine regulation of elongation of long-chain fatty acids family member 3 and 6 (ELOVL3 and ELOVL6) expression in primary human keratinocytes.

Keratinocytes were differentiated in the absence or presence of IL-4/IL-13. Keratinocytes were transfected with control siRNA, STAT6 siRNA, ELOVL3 siRNA, ELOVL6 siRNA, or ELOVL3/ELOVL6 siRNA and underwent Ca²⁺ differentiation for the period of time indicated. The expressions of STAT6 mRNA (A and D), ELOVL3 mRNA (B and E), and ELOVL6 (C and F) were analyzed. (G) ELOVL3 immunostaining in keratinocytes. ELOVL3 staining, yellow; DAPI, cell nuclei staining, blue. Magnification, 250×. *P < 0.05, **P < 0.01, 2-tailed Student’s t test (n = 3).