Integrative lipidomic features identify plasma lipid signatures in chronic urticaria

Abstract

Chronic urticaria (CU) is a chronic inflammatory skin disease mainly mediated by mast cells. Lipids exert essential functions in biological processes; however, the role of lipids in CU remains unclear. Nontargeted lipidomics was performed to investigate the differential lipid profiles between CU patients and healthy control (HC) subjects. Functional validation studies were performed in vitro and in vivo including β-hexosaminidase release examination from mast cells and passive cutaneous anaphylaxis (PCA) mouse model. We detected dramatically altered glycerophospholipids in CU patients compared with HCs. Phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) were increased, while phosphatidylcholine (PC) was reduced in CU patients. The reduction in PC was related to a high weekly urticaria activity score (UAS7), while PS was positively associated with the dermatology life quality index (DLQI). We also identified the differential lipid profiles between chronic spontaneous urticaria (CSU), symptomatic dermographism (SD), and CSU coexist with SD. CU patients were classified into two subtypes (subtype 1 and subtype 2) based on consensus clustering of lipid profiling. Compared with patients in subtype 2, patients in subtype 1 had elevated levels of PC (18:0e/18:2) and PE (38:2), and lower urticaria control test (UCT) scores indicated worse clinical efficiency of secondary generation H1 antihistamines treatment. Importantly, we found that supplementation with PC could attenuate IgE-induced immune responses in mast cells. In general, We described the landscape of plasma lipid alterations in CU patients and provided novel insights into the role of PC in mast cells.

Keywords: chronic urticaria; glycerophospholipid; lipidomic signatures; mast cell; phosphatidylcholine.

Figure 1

Identification of lipid profiling in plasma of CU. (A) PLS-DA score plots show discrimination between the HC (blue) and CU (red). (B) Validation plots were obtained from 200 permutation tests (R2 = 0.672, Q2 = 0.365). (C) Volcano map of differentially expressed lipids between CU and HC groups. Blue represents the down-regulated lipid molecules, orange represents the up-regulated lipid, and non-significant lipid molecules are gray. Molecules, solid and round represent the lipid molecules VIP ≥1, hollow and round represents the lipid molecules VIP < 1. (D) Heatmap of the top 7 differentially lipids between HC and CU. Each row in the figure represents a differential lipid, and each column represents a sample. Different colors indicate different intensities, and colors range from blue to red, indicating strength from low to high. (E) Lipid metabolites set enrichment analysis for CU. Redder colors represent lower P values, and larger circles represent higher enrichment ratios. Low P-value and large enrichment ratio indicate that this pathway is enriched more. (F) The correlation analysis of different lipids showed that the same lipids had a good correlation.

Figure 2

Alterations in lipids are related to the clinical characteristics of CU. Heatmaps of lipid clusters grouped by different UAS7 (A) and DLQI (B) scores are shown on the left. The abscissa marks the specific scoring groups, and the ordinate marks the cluster and the major lipids in that cluster. The cut squares in the figure correspond to groups and clusters. Clusters with significant trends are on the right.

Figure 3

Identification of Lipid signatures in subtypes of CU. (A-C) Comparative analysis of lipid abundance between three groups of different types of urticaria as shown in the volcanic diagram. Blue represents the down-regulated lipid molecules, orange represents the up-regulated lipid, and non-significant lipid molecules are gray. (D) Heatmap of the top 15 differentially expressed metabolites. Different colors indicate different intensities, and colors range from blue to red, indicating strength from low to high. (E) Boxplot of the 15 significantly altered lipids. Blue represents the HC, yellow represents the CU, orange represents the SD, and purple presents the CSU+SD. The y-axis is the normalized intensity after log2 transformation.

Figure 4

Classification of CU lipid subtypes based on profiling. (A) The heat map corresponding to the consensus matrix for 2 metabolite subtypes was obtained by applying consensus clustering. (B) CU was classified into two subtypes based on lipid profiling, subtype 2 shared higher UCT scores compared with subtype 1. (C) The heatmap of all differential lipids related with subtype 1 (blue) and subtype 2 (orange), which was generated using the heatmap function in R with subtypes, UAS7 (green), DLQI (purple) score and UCT (blue) score as the annotations. The colors range from blue to red, indicating strength from low to high. (D, E) Plasma PC (18:0e/18:2) (R=-0.28, P-value=0.041) and PE (38:2) (R=-0.29, P-value=0.038) levels were negatively correlated with UCT scores.

Figure 5

Supplementation with PC attenuates IgE-induced mast cell activation in vitro and in vivo. (A) The mean plasma PC level of the CU patients n = 42 was significantly lower than that of the HC subjects n = 42 were determined by ELISA. (B) PC (Including three concentrations: 1 nM,10 nM, and 100nM) inhibited the degranulation of RBL-2H3 cells and BMMCs, and there was no concentration dependence. (C) Bodyweight curve and phenotypic changes of four mice groups (Control, n = 5; PC, n = 5; IgE+DNP-HSA, n = 8; IgE+DNP-HSA+PC, n = 8) during the experiment. (D) PC intervention reduced skin vascular permeability in the PCA mice model. (E) Toluidine blue staining showed that PC inhibited degranulation of skin mast cells. (F) The expression of genes involved in cytokine was measured by real- time-qPCR analysis. PC inhibited cytokine release in PCA mice models, including IL-4, IL-10, IL-13, CCL-2, CCL-3, and TNF- α. The data from multiple experiments were expressed as the mean ± standard deviation (SD). Significant differences were evaluated using student t-test or the nonparametric test (Be denoted as follows: ****p≤.0001, ***p ≤.001, **p ≤.01, *p ≤.05).

Figure 6

Integrative lipidomic features identify plasma lipid signatures in chronic urticaria. The lipidomics was performed to investigate the differential lipid profiling between CU patients and healthy control subjects (HC). The levels of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) in CU patients were significantly increased, while those of phosphatidylcholine (PC) were significantly decreased. The decrease of PC was highly correlated with urticaria activity score (UAS7), while PS was positively correlated with dermatological quality of life index (DLQI). Differences in lipid profiles between CSU, SD, and CSU+SD. CU patients were divided into two subtypes, subtype 1 and subtype 2, based on consistent clustering of lipid profiles. Compared with subtype 2, subtype 1 was characterized by increased PC (18:0E/18:2), increased PE (38:2), and lower UCT score, indicating poor clinical efficacy of second-generation H1 antihistamine therapy. PC supplementation attenuated IgE - induced mast cell immune responses in the passive cutaneous anaphylaxis model. This figure is created with https://BioRender.com.