PV can cause multisystem damage as well as skin damage. Research has highlighted the vital roles of adaptive immune molecules, such as IL-17, IL-23, and TNF-α (1), in PV, which has allowed the development of multiple effective therapies targeting tumour necrosis factor-alpha (TNF-α; adalimumab), IL-17A (secukinumab), IL-17 receptor A (IL-17RA; brodalumab), and IL-23 (guselkumab) (2–4). Among them, secukinumab and adalimumab are the two most widely used biological agents in China. These biological agents have significantly improved the efficacy of psoriasis treatment (5), but a loss of effectiveness (6) or the occurrence of phenotypic transformation (7) is not uncommon in clinical practice. These phenomena suggested that using biological agents might alter immune pathology and highlight the pleiotropic nature of these molecules. Further investigations are needed to explain these phenomena.

Recent developments in the field have moved towards delivering high-quality and consistent quantification in large-scale projects. DIA-MS is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy (8). DIA-MS has been successfully used for the investigation of skin tumours (9) and atopic dermatitis (AD) (10). Meng et al. used DIA-MS to identify serum proteins distinguishing responders from nonresponders to an oral traditional Chinese medicine to explore diagnostic and predictive disease biomarkers in serum (11), and they demonstrated the clinical utility of an in-depth serum proteomic platform to identify specific diagnostic and predictive biomarkers of psoriasis. These results suggest that DIA-MS can be used to study the skin of patients with psoriasis.

A few studies have provided significant insights into the changes in proteins or genes in psoriatic lesions after treatment with biological agents. Kolbinger et al. quantified the levels of 170 proteins in patients with psoriasis before and after administration of the secukinumab. The results showed that many dysregulated immune molecules returned to normal after treatment with secukinumab (12). Foulkes et al. investigated the serum proteome of patients with severe psoriasis treated with the TNF inhibitor etanercept. The study found that the baseline serum proteome can indicate patient response to biological therapies (13). However, the platforms used in these previous studies have limited ability to detect protein species. Therefore, the characterization of the change in proteins in the skin after treatment with biological agents through DIA-MS is an attractive possibility.

Here, we used DIA-MS to delineate the proteomic landscape of the skin of patients with moderate to severe psoriasis. In summary, from a posttreatment point of view, these data identified IL-17A and TNF-α as critical molecules that maintain psoriasis plaques. The inhibition of these molecules can induce massive protein changes over time and have overlapping influences on immune response and process through direct or indirect effects.

Here, we reported a remarkable efficacy of biological agents in psoriasis, which is consistent with previous studies (2, 5). Our research verified that the inhibition of IL-17A and TNF-α can induce multiple molecular changes in psoriatic lesions and have overlapping influences on immune responses and processes, such as different kinds of signalling pathways on immune responses and processes, through direct or indirect effects ( Figures 3B , 4B , 5 , 7C , 8 ).

We quantified up to 6054 proteins through the use of DIA-MS, the development of which was a major advance for global protein quantification across multiple samples. In our research, the FC of DEPs was significantly lower than that in a previously reported transcriptomic study (19) because proteins are the final effector molecules in biological systems, while significant differences in their corresponding mRNA levels can exist (20, 21).

Our data revealed new insights into molecular changes in psoriatic skin after the inhibition of IL-17A and TNF-α and identified some important DEPs that participate in immune regulation and biological processes. For example, heat shock protein HSP 90-beta (HSP90AB1) is involved in multiple pathways and processes of cells (22) and is vital for IL-17A-mediated signalling. A previous report found that the HSP90 inhibitor RGRN-305 significantly reduced the IL-17A- and TNF-α- induced proinflammatory genes in human keratinocytes in vitro (23). In our study, HSP90 was downregulated after biological treatment and was positively correlated with the PASI score. Signal transducer and activator of transcription 1 (STAT1) was also downregulated; STAT1 mediates cellular responses to interferons (IFNs) and cytokines, acts as a suppressor of NF-κB (24) and synergizes with IL-17. In addition, critical downstream molecules, including S100A7, S100A8, and S100A9, act as antimicrobial peptides and chemotactic factors and play an essential role in innate immunity. Studies have shown that these proteins are associated with disease severity in psoriasis patients (25, 26). In our study, they were all downregulated which indicated a suppression of the immune response and a reduction in disease severity after treatment in both biological agent groups.

Although the drug targets were entirely different, some DEPs were involved in many common immune pathways or biological processes, such as LXR/RXR activation, acute phase response signalling, the MSP-RON signalling pathway, the role of IL-17A in psoriasis, and IL-12 signalling and production in macrophages. We found that eleven proteins (PLXDC2, IMPDH2, ALCAM, HSP90AB1, GAA, COL3A1, PCNA, LGALS3, MATN2, NAMPT, and CBR4) were altered in both groups, and their expression tended to be the same. For instance, galectin 3 (LGALS3) and CD166 (ALCAM) were upregulated in both groups. Research has shown that LGALS3 and CD166 can both bind to CD6. LGALS3 can interfere with the events induced by the CD6-CD166/ALCAM adhesive interactions, followed by T-cell proliferation and cell adhesion (27). Itolizumab, an antibody to CD6, has been applied to treat moderate to severe psoriasis and is effective and well tolerated (28). These proteins were analysed by IPA, and the results showed that they can affect some important pathways, such as the TNF, PI3K-AKT, ERK, NF-κB (complex), IL-17R, MAPK, and interferon-alpha signalling pathways, by direct or indirect action. Therefore, they may be a focus for further study in psoriasis.

Nograles KE et al. found that IL-17 and IL-22 mediate distinct downstream pathways that contribute to psoriatic phenotype (29). Krueger JG et al. used gene set variation analysis (GSVA) to reveal changes in many cell type-specific and inflammatory genes and pathways important in psoriasis (19) after secukinumab therapy. The same results were found in studies by Foulkes AC et al. (13) and Xu M et al. (11) at the serum proteome level after treatment with etanercept and traditional Chinese medicine, respectively. In our research, IPA canonical pathway analysis showed that the separate pathways in the adalimumab group were mainly related to metabolism, such as arginine degradation and oxidative phosphorylation, while in the secukinumab group, the separate pathways primarily focused on the immune system

A previous study showed that an IL17A inhibitor has a neutral impact on metabolic parameters (30), but another study identified changes in genes that regulate peripheral lipid metabolism (19). In our study, metabolic pathways were not activated in the secukinumab group, although some proteins were involved. The reason for the inconsistent results may be the different detection specimens and methods. Therefore, further study is needed to determine whether biological therapy improves the metabolic status and metabolic complications of patients.

Our study also found few DEPs enriched in keratinization that were associated with keratinocyte function and the immune response. Moran et al. found that correcting the dysregulated Th17:Treg axis in HS patients treated with adalimumab (31) provided a rationale for targeting IL-17 in this inflammatory keratosis disease (32). In addition, our data provide clues to phenotypic transformation during biological treatment. For example, kallikrein 5 (KLK5) is overexpressed after secukinumab treatment. KLK5 is a serine protease involved in cell renewal and regulation of skin barrier function (33), and abnormal KLK-5 expression and the hyperactivation of KLK-5 are known to induce atopic dermatitis-like lesions in the skin (34). Some important proteins involved in keratinization, such as KRT6 and KRT17, which have been reported to play a role in psoriasis in previous studies using the IMQ-psoriasis mouse model (35), were not identified in this study. We speculate that this may be due to the different intervention drugs, models and detection methods used.

Studies have found that TNF blockade does not affect or even promote the protein levels of IL-17 (36, 37). Another study found limited effects on alterations in TNF levels in patients treated with secukinumab (38). Krueger et al. found that IL-17A, in synergy with TNF, induces inflammatory products in KCs and ultimately representative psoriatic plaques (19). These findings suggested that the reduced skin inflammation observed in patients after blocking IL-17A is independent of changes in TNF levels and vice versa. Considering that the relevant cytokines and their changes were not detected in this study, we cannot make a direct judgement on this. Nevertheless, we identified an overlapping effect among some proteins on signalling pathways and immune response processes at the proteomic level. Furthermore, it has been shown that bispecific anti-TNF-α/IL-17 antibodies may have superior efficacy and safety in treating rheumatoid arthritis (39), but this has not been demonstrated in another study involving patients with psoriasis arthritis (40). This may be due to partly different pathogenesis between them. Therefore, we suggest that in treating psoriasis, the co-use of these inhibitors may achieve a better effect than the use of a single agent.

This study did not identify two well-known cytokines that play an essential role in the pathogenesis of psoriasis and as drug targets (TNF-α and IL-17A). Similar results were found in another serum proteomic study, in which the authors attributed the lack of the identification of these cytokines to the pathophysiologic heterogeneity of psoriasis disease in the population (40). We think that the lack of detection of these cytokines in DIA and PRM analyses may be due to the limitation of DIA for detecting low-abundance proteins (41). To overcome this limitation, future research would fuse targeted proteomics (such as MS3-based methods) or commercial protein microarrays with DIA to reliably identify high- and low-abundance proteins.

(1) The sample size was relatively small. (2) Some vital low-abundance proteins were not detected because of limited detection ability by DIA. (3) The median age and course of the disease were lower in the adalimumab group ( Table 1 ), so their impact on our data interpretation could not be precisely defined. To a certain extent, these confounding factors might influence the research results.

In conclusion, this study presents a systematic proteomic investigation of skin samples from two biological agent groups with different mechanisms of action and a control group of psoriasis patients. The results indicated that inhibiting these molecules could induce multiple molecular changes in psoriatic lesions and have overlapping influences on immune responses and processes, such as different kinds of signalling pathways and immune processes, through direct or indirect effects. Our results suggested that the combined use of these two inhibitors may provide better efficacy in treating psoriasis.

The data presented in the study are deposited in the iProX repository (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD037579), accession number PXD037579.

The studies involving human participants were reviewed and approved by Dermatology Hospital of Zhejiang Province (LL-2020-15). The patients/participants provided their written informed consent to participate in this study.

QD and DL designed and supervised the project. QD wrote the manuscript with input from the authors. BBX, XXJ, YLT, GHL, NNS, and XBY. Collected the samples and clinical data. LHH and JH preserved the samples. All authors contributed to the article and approved the submitted version.

The authors thank Genechem Co., Ltd. (Shanghai, China) for the mass spectrometric analysis. We thank Dr Sun. for data computation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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