3.1 Non-target metabolomics reveals the metabolic profiling of psoriasis patients before and after Ixekizumab treatment
In the first step, metabolic profiling analysis was conducted in the study cohort 1 including healthy people (CON), psoriasis patients (PSO) and Ixekizumab treated patients (IXE). After raw data filtering and simple processing, processed data were carried out on PCA. Whether in positive ion or negative ion mode, IXE group was invariably between the CON and PSO groups, which indicated the metabolic profiling of Ixekizumab-treated psoriasis patients converted into normal status (Fig. S1a, b). To better investigate the abnormal metabolites caused different metabolic profiles, OPLS-DA was using in following PSO/CON and IXE/PSO paired-comparisons (Fig. S1c-f).
According to the constructed multivariate models, in conjunction with univariate statistical analysis, 37 differential metabolites were revealed in PSO/CON comparison (Fig. 2) and 31 differential metabolites were accurately identified in IXE/PSO comparison (Fig. S2). In total of 43 metabolites including lysophospholipids (LPLs), free fatty acids (FFAs), dicarboxylic acids (DAs) and acyl-carnitines that contributed most to the differentiation of groups was identified in the study cohort 1 (Table S1).
Receiver operating characteristic (ROC) curve analysis is generally considered to be the gold standard for the assessment of biomarkers performance. Given that area under the curve (AUC) of ROC curves was high than 0.6, these identified metabolites could be regarded as candidate biomarkers in the Ixekizumab treated psoriasis patients (Fig. S3).
3.2 IL-17A monoclonal antibody ameliorate dysregulated lipids metabolism in psoriasis patients
As visualized in the heat map, LPLs and FFAs were upregulated in psoriasis patients compared with healthy people, while DAs and acyl-carnitines were altered in the opposite way (Fig. 3a). Lysophosphatidylcholines (LPCs), lysophosphatidylinositols (LPIs) and lysophosphatidic acids (LPAs) were significantly upregulated in the psoriasis patients. It has been demonstrated that the circulating LPCs and LPAs have the potent pro-inflammatory effect and raised in the several inflammation-associated diseases including psoriasis[18, 19]. Moreover, the presence of n-6 polyunsaturated fatty acids on LPCs have the stronger ability to evoke inflammatory response. In this study, LPC (22:5) and LPC (20:3) with n-6 polyunsaturated fatty acids had highest fold-change values among all the identified LPCs (Fig. 3b). In accordance with the increased levels of LPCs, as its downstream product, glycerophosphocholine (GPC) level was also explosively upregulated in psoriasis patients (Fig. 3b). As LPCs can be produced by phosphatidylcholines, decreased levels of phosphatidylcholines further proved disordered phospholipids pathway. In addition to LPCs, another LPLs, LPIs were also upregulated in psoriasis patients (Fig. 3b). Although LPIs have relatively lower concentration in human blood compared with LPCs, they have abundant biological functions including pro-inflammatory.
In the Land’s cycle, FFAs can be produced from LPLs by phospholipase A . As expected, FFAs levels were likewise higher in the serum of psoriasis patients. Compared with upregulated FFAs, DAs and acyl-carnitines decreased in psoriasis patients (Fig. 3b). Since DAs were the intermediates in the ω-oxidation pathway and acyl-carnitines were the “vehicles” in the β-oxidation process, decreased levels of these two metabolites indicated potential dysfunction of fatty acids decomposition and in turn result in higher blood levels of FFAs. Accumulation of FFAs has been reported to constantly sensitize dendritic cells to amplify Th1/Th17 immune responses , which was supported by the inverse correlation between DAs levels and psoriasis area and severity index (Fig. 3c).
After Ixekizumab treatment, the most obvious metabolic changes in psoriasis patients were the decreased LPCs and GPC. Especially the aforementioned inflammation-associated LPC (20:3) and LPC (22:5) were drastically decreased in the treated patients. With the decrease of LPCs, PCs increased synchronously and returned to the normal levels together. The downstream product of LPCs, GPC showed the most decreased trend among all the differential metabolites, which could be explained by the decreased LPCs. Average levels of acyl-carnitines and FFAs were ameliorated at the same time although not statistically significant. However, DAs were upregulated to normal levels in treated patients. These results highlighted that the treatment of IL-17A mAb might not only ameliorate the lesions of psoriasis, but also restore the dysregulated lipids metabolism to normal level in psoriasis patients.
3.3 Common and specific metabolites in psoriasis patients with or without cardiovascular comorbidities
Out of the 43 identified metabolites in study cohort 1, 18 differential metabolites overlapped with previously reported potential biomarkers of CVDs. To validate the correlation of the identified differential metabolites and CVDs, the second study cohort was recruited with additional groups of psoriasis patients with coronary heart diseases (PC) and coronary heart diseases patients without psoriasis (CV). Considering psoriasis patients with cardiovascular comorbidities were mainly elderly and age was the critical factor in the CVDs, additional healthy people (CON) and psoriasis patients (PSO) of comparable age were also recruited in the study cohort 2.
As expected, the results of metabolomics analysis on the study cohort 2 largely conform to the previous observations in study cohort 1. In sPLS-DA, PC group was between PSO and CV groups and apparent separation was achieved among the four groups (Fig. 4a). 25 differential metabolites identified in PSO/CON comparison in the study cohort 1 were demonstrated to be significantly as well in the PSO, CV and PC groups comparing to CON group in the study cohort 2 (Fig. 4b, c). Although all identified LPLs were upregulated in PSO and PC groups of two study cohorts, LPCs levels were even higher in PC group compared PSO group (Fig. 4d). In the study cohort 2, the aforementioned LPC (22:5), LPC (20:3) and GPC not only drastically increased in PSO group, but also had higher levels in PC group. Although all the identified LPIs upregulated in the PSO group of two study cohorts, psoriasis patients with coronary heart diseases had significant lower levels (Fig. 4d).
The dysregulated trends of DAs in patients of study cohort 2 were also consistent with the results of study cohort 1, but there were no significant changes between PSO and PC groups (Fig. 4c, d). Considering that DAs levels were inversely correlated with psoriasis area and severity index in study cohort 1, this outcome was beyond our expectations. The results of metabolic profiling in study cohort 1 had demonstrated acyl-carnitines, a critical carrier in fatty acid oxidation, decreased in the psoriasis patients. In study cohort 2, although middle-chain acetyl-carnitines decreased in all patients, no significant changes were found between PSO and PC groups, which was consistent with the results of DAs.
Although only six individuals in PC group were treated with Ixekizumab, the changes of lipids metabolism conformed to the previous observations in study cohort 1. After Ixekizumab treatment, LPLs, DAs and acyl-carnitines were ameliorated to normal levels in psoriasis patients with coronary heart diseases (Fig. 5). This result indicated that IL-17A mAb might restore the dysregulated lipids metabolism to normal level in psoriasis patients with coronary heart diseases.