Clinical Significance of Decreased TIPE2 Expression in Peripheral Blood Mononuclear Cells of Patients with Psoriasis Vulgaris

Psoriasis is an immune system disorder induced by the interaction of the polygenic background and environmental factors. Tumor necrosis factor-α (TNF-α )-induced protein 8-like 2 (TNFAIP8L2, TIPE2), a regulator of immunity, is diferentially expressed in tumors, infammation


Introduction
Psoriasis is an immune-mediated infammatory disease primarily afecting the skin and joints, with an overall incidence ranging from 0.14% in East Asia to 1.99% in Western Europe [1]. In 2014, the World Health Organization defned psoriasis as a chronic and noncommunicable disease that leads to pain, disfguration, and disability [2], posing substantial fnancial and psychological burden on individuals and society. Moreover, the risks of concurrent metabolic syndrome (hypertension, obesity, dyslipidemia, and type 2 diabetes mellitus), cardiovascular disease, and malignancy among patients with psoriasis are rising [3].
Psoriasis vulgaris is the most prevalent type, afecting approximately 90% of patients with psoriasis. Typical skin lesions are well-demarcated erythematous plaques covered with a thick white micaceous scale; removal of the scale leads to punctate bleeding (Auspitz sign) [4]. Te pathogenesis of psoriasis is complicated, and the primary cause is an immune system disorder induced by the interaction of the polygenic background and environmental factors (infection, use of medication, stress, surgery, smoking, and alcohol abuse). Te immune mechanisms associated with T helper (T) 1, T17, and T22 are essential in psoriasis development. In the initial stage of psoriasis, plasmacytoid dendritic cells, keratinocytes, natural killer T cells, and macrophages secrete cytokines such as TNF-α, interferon (IFN)-c, interleukin (IL)-1, and IL-6 to activate myeloid dendritic cells (mDCs) [5]. Subsequently, mDCs migrate to the lymph nodes and secrete IL-23 and IL-12, inducing naive CD4+ T cells (T0) to diferentiate into T1, T17, and T22 cells. T1 cells secrete TNF-α and IFN-c, T17 cells secrete IL-17, IL-22, and TNF-α, and T22 cells secrete IL-22 [6]. Tese congenital and adaptive immune cells and their secreted cytokine networks are collectively involved in the initiation and development of psoriasis, providing new insights into its treatment.
TIPE2 is a recently discovered negative regulator involved in immunological homeostasis [7]. Mice lacking TIPE2 manifest chronic infammatory diseases at approximately 2 months old, characterized by weight loss, splenomegaly, leukocytosis, multiple organ infammation, and elevated levels of cytokines such as TNF-α, IL-1, IL-6, IL-10, and IL-12. Experiments in vitro revealed that TIPE2 could bind to caspase-8 and inhibit activation of activator protein-1 and nuclear transcription factor-κB (NF-κB) [7]. In addition, current studies indicated that the expression of TIPE2 is signifcantly downregulated in patients with infectious and autoimmune diseases such as chronic hepatitis B, asthma, primary biliary cirrhosis, myasthenia gravis, and systemic lupus erythematosus [8][9][10][11][12]. Tis evidence suggests that TIPE2 presumably plays an essential role in infammatory and especially autoimmune diseases by serving as a negative regulator of immunity.
Te normal function of TIPE2 is paramount for immune homeostasis, and existing studies have shown that TIPE2 expression is closely correlated with multiple autoimmune diseases. However, TIPE2 expression and its potential implications in psoriasis vulgaris remain unclear. Terefore, this study aimed to detect the TIPE2 mRNA expression levels in the PBMCs of patients with psoriasis vulgaris and analyze the correlation between the levels of TIPE2 and other infammatory cytokines in psoriasis vulgaris pathogenesis.

Human Subjects.
Tis case-control study enrolled 33 patients who were frst treated in the Department of Dermatology and Venereology at the First Afliated Hospital of Xinxiang Medical University from October 2021 to March 2022-they were clinically confrmed to have psoriasis vulgaris. Patient clinical data, including age, sex, disease stage, body mass index (BMI), family history, history of respiratory infection, allergy history, and other baseline information, were recorded. Te severity of psoriasis was assessed using the PASI score.
Patients with other types of psoriasis (pustular, erythrodermic, guttate psoriasis, and psoriatic arthritis), other skin diseases, tumors, history of blood disorders, acquired immune defciency syndrome, infammatory bowel disease, or other immunosuppressive diseases were excluded from the study. Additionally, we excluded patients with psoriasis vulgaris who had taken any antipsoriatic medication (topical or systemic) within the past month or had undergone biological therapy within the last 6 months. During the same period, 31 healthy subjects without a history of chronic dermatological or systemic diseases were recruited as the control group from the Physical Examination Center at our Hospital. Te study was approved by the Ethics Committee of the First Afliated Hospital of Xinxiang Medical University (NO 2020045). All the participants signed informed consent forms.

RNA and cDNA Preparation from PBMCs.
Heparinized venous peripheral blood (5 mL) was collected from each subject and stored at 4°C for no more than 4 h. Tereafter, an equal volume of washing bufer (Solarbio, Beijing, China) was added for dilution. PBMCs were isolated from the peripheral blood using Ficoll density gradient centrifugation (Solarbio). Total RNA was extracted from unstimulated PBMCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of RNA was reversetranscribed into cDNA using a frst-strand cDNA synthesis kit (Novoprotein, Shanghai, China).

Real-Time Quantitative PCR (qPCR)
. qPCR was performed using a QuantStudio DX Real-time PCR Instrument (Termo Fisher Scientifc, Waltham, MA, USA). Te primers used for qPCR are listed in Table 1. qPCR was performed using SYBR Green qPCR SuperMix Plus (Novoprotein) according to the manufacturer's instructions. Te reaction conditions for the qPCR were denaturation at 95°C for 1 min, followed by 40 cycles at 95°C for 20 s and 60°C for 1 min. All the samples were evaluated thrice. Te β-actin gene was used as an internal control. Results were determined using the comparative (2 −△△Ct ) method.

Statistical Analysis.
All statistical analyses were performed using the SPSS version 26.0 (SPSS Inc., Chicago, IL, USA). Descriptive data are expressed as mean (standard deviation (SD)), median (interquartile range (IQR)), and percentage (%). Chi-squared test was used to analyze the classifed variables, and the independent samples t-test was applied to compare age and BMI means. Te diferences among TIPE2, RELA, TNF-α, IL-10, IL-6, and IL-1β mRNA expression levels between patients with psoriasis vulgaris and healthy controls were tested using the Mann-Whitney U test. Te Mann-Whitney U test was used to evaluate the diferences in mRNA expression levels of cytokine and clinical characteristics. Te correlation between cytokine levels in the PBMCs of patients with psoriasis vulgaris and the PASI score and the expression levels of TIPE2 and other infammatory cytokines were analyzed using Spearman's correlation analysis. P < 0.05 was considered statistically signifcant.

Expression Levels of TIPE2 and Other Infammatory Cytokines in Diferent Stages of Patients with Psoriasis
Vulgaris. Here, we investigated the diferential expression of cytokines in patients with psoriasis vulgaris at diferent stages. It was demonstrated that TIPE2 mRNA expression in the active stage was higher than that in the stationary stage   Figure 3).

Te Predictive Value of TIPE2 Expression Level in Patients with Psoriasis
Vulgaris. In addition, we used the receiver operating characteristics (ROC) analysis to evaluate the ability of TIPE2 mRNA to diferentiate between patients with psoriasis vulgaris and healthy controls, as well as between the active and stationary stages of psoriasis vulgaris. Figure 4(a) showed that the area under the ROC (AUROC) curves for TIPE2 mRNA in predicting the psoriasis vulgaris was 0.673 (95% CI 0.535-0.810, P � 0.018), and the optimal cut-of level was 1.211 with a sensitivity of 81.82% and a specifcity of 61.29%. Furthermore, Figure 4(b) shows the AUROC of TIPE2 mRNA in predicting the active stage in patients with psoriasis vulgaris was 0.745 (95% CI 0.548-0.942, P � 0.04), with optimal cut-of level of 0.877 and a sensitivity of 100% and a specifcity of 44%.

Diferential Cytokine Expression among Other Clinical
Characteristics of Patients with Psoriasis Vulgaris. To explore whether the clinical characteristics afect the expression levels of cytokines in the PBMCs of patients with psoriasis vulgaris, including sex, family history, allergy, and respiratory infection history, we compared and analyzed the relationship between the expression levels of cytokines and these factors. As shown in Table 3, higher TNF-α mRNA expression in PBMCs was observed in patients with psoriasis vulgaris with a history of respiratory infection than that in those without it (2.682 (IQR 1.537-3.321) versus 1.349 (IQR 0.7-2.316), P < 0.05). Tere were no signifcant diferences among the sex, family history, and allergy history subgroups.

Association between TIPE2 mRNA Expression and Other Infammatory Cytokines in Patients with Psoriasis Vulgaris.
Furthermore, we performed a correlation analysis between TIPE2 and other infammatory cytokine levels. Te results revealed that TIPE2 mRNA expression positively correlated with RELA (r � 0.509, P � 0.002) and TNF-α levels (r � 0.7, P < 0.001) and negatively correlated with IL-6 (r � −0.372, P � 0.034). No relationship was found between the levels of TIPE2 and IL-10 (r � 0.181, P � 0.312) and IL-1β (r � 0.139, P � 0.438) in the PBMCs of patients with psoriasis vulgaris ( Figure 5).

Discussion
Infammation and immune dysfunction are critical to the pathogenesis of psoriasis [4]. TIPE2 is indispensable for infammation regulation and maintenance of immunity [7], but its exact efects on the development of psoriasis remain unclear. Tis case-control study determined the TIPE2 mRNA expression level in the PBMCs of patients with psoriasis vulgaris. To our knowledge, this is the frst clinical study to investigate this. TIPE2, a cytosolic protein, was initially obtained from mouse models of experimental autoimmune encephalomyelitis. It is preferentially expressed by lymphocytes and macrophages but may be induced in other cell types by TNF-α. TIPE2 can prevent excessive immune responses and  4 Dermatologic Terapy sustain homeostasis in the immune system [7]. TIPE2 −/− cells have hyperreactivity toward toll-like receptor and T-cell receptor (TCR) activation, and TIPE2 suppresses NF-κB activation [7] in T cells and macrophages and induces macrophage polarization to an M2 phenotype [14]. Recent studies have shown that TIPE2 is essential in the occurrence and development of tumors, infammation, and autoimmune diseases. Te level of TIPE2 expression is signifcantly weak in patients with primary hepatocellular carcinoma, exhibiting a negative correlation with tumor migration and invasion [15]. Furthermore, decreased TIPE2 expression has also been found in nonsmall cell lung and gastric cancers, TIPE2 overexpression could ultimately inhibit the invasion, migration, and metastasis of tumor cells [16,17]. However, in renal cell carcinoma cells and tissues, the expression of TIPE2 is prominently increased and is positively correlated with TNM staging [18]. Except for tumors, TIPE2 expression levels are signifcantly reduced in the PMBCs of patients with chronic hepatitis B and C and negatively correlated with ALT and AST levels and virus DNA load [8,19]. A recent study indicated that TIPE2 defciency increased the susceptibility of corneas to Pseudomonas aeruginosa infection and worsened keratitis by enhancing NF-κB signaling and infammatory cell infltration [20]. As for autoimmune diseases, TIPE2 expression is decreased in PBMCs from patients with asthma, primary biliary cirrhosis, and myasthenia gravis and is negatively correlated with the expression of key proinfammatory cytokines [9][10][11]. Te expression of TIPE2 in the PMBCs of systemic lupus erythematosus patients was also found to be signifcantly decreased and negatively correlated with the SLE disease activity index [12]. However, signifcantly increased levels of TIPE2 expression were found in the PBMCs of patients with ankylosing spondylitis and rheumatoid arthritis [21,22]. We found that the TIPE2 mRNA expression levels in the PBMCs of patients with psoriasis vulgaris were markedly lower than those in healthy volunteers. In contrast, the expression levels of other infammatory cytokines increased, illustrating that TIPE2 defciency might be a predisposing factor for the initiation and development of psoriasis. Interestingly, TIPE2 mRNA expression negatively correlated with IL-6 expression and positively correlated with TNF-α expression. In addition, TIPE2 mRNA expression was higher in the active stage than in the stationary stage. TIPE2 serves as a negative regulator of infammation and immune homeostasis. We speculate that early in the progression of psoriasis, compensatory secretion is induced in T cells or macrophages by TNF-α, regulating immune and infammatory processes by suppressing NF-κB activity. Our research found that a cut-of level of 0.877 for TIPE2 mRNA expression demonstrated potential in discriminating active stages of psoriasis vulgaris from stationary stages, but the AUROC curves for TIPE2 mRNA in predicting the psoriasis vulgaris was small to have limited accuracy. Tis suggested that TIPE2 could serve as a potential diagnostic tool for early detection of active stages of psoriasis vulgaris. However, in order to validate this hypothesis, further investigation utilizing a larger and prospective cohort is warranted. Decreased expression of TIPE2 may lead to elevated proinfammatory cytokine levels in patients with psoriasis vulgaris. Our results showed that the expression levels of RELA, TNF-α, IL-10, IL-6, and IL-1β in the PBMCs of patients were signifcantly higher than those in healthy controls, presenting a positive relationship with the PASI score. Proinfammatory cytokines such as TNF-α, IL-6, and IL-1β participate in the pathogenesis of multiple infammatory diseases. TNF-α is critical in psoriasis pathogenesis as it promotes T17 cell diferentiation by activating mDCs to produce IL-23. Te TNF-α inhibitor was the earliest applied biological agent for psoriasis with good efcacy, implying the importance of TNF-α in promoting and maintaining the disease [23]. Furthermore, analysis of the expression profle of genes associated with transforming growth factor β (TGFβ) signaling demonstrated that anti-TNF drugs appeared to have a greater efect on TGFβ cascades than that of cyclosporine A [24]. Verma et al. [25] and Arican et al. [26] reported results consistent with our fndings that serum TNF-α, IL-6, and IL-1β levels in patients with psoriasis were signifcantly higher than those in the control group. Furthermore, Cataldi et al. [27] acquired similar results for serum TNF-α, IL-6, and IL-1β. Evidence suggests that IL-10 can inhibit the synthesis of other infammatory cytokines as a negative immunomodulator, which may be vital in psoriasis [28]. We found that IL-10 mRNA expression was signifcantly elevated in the PBMCs of patients with psoriasis vulgaris, consistent with previous studies [27]. However, Kutwin et al. [29] reported the opposite; patients with psoriasis had signifcantly reduced IL-10 mRNA expression in their skin lesions. Te discrepancy in these results might be due to the diferent determination methods and study designs in each study. Considering these contradictory results, the role of IL-10 in psoriasis warrants further investigation.
Environmental factors, especially infections, have been revealed to play important roles in the predisposition and exacerbation of psoriasis [30]. A large cohort study of a Dutch population reported that the risk of severe infections in patients with psoriasis was signifcantly higher than that in control subjects. Respiratory, skin, and abdominal infections commonly occur in patients with psoriasis [31]. Another study from Britain proposed that psoriasis was related to an increased risk of severe infection, and patients with moderate or severe psoriasis exhibited a higher risk; respiratory infection was the most common type of infection among patients with psoriasis [32]. We found that patients with psoriasis vulgaris with a history of respiratory infection had higher TNF-α mRNA expression in their PBMCs than that in those without an infection history, implying a link between respiratory infection and psoriasis vulgaris, in which TNF-α is a crucial factor.
Te mechanisms involved in the downregulation of TIPE2 mRNA expression levels in the PBMCs of patients with psoriasis vulgaris remain unclear. It has been revealed that TIPE2 expression is signifcantly downregulated, while expression of MicroRNA (miR)-21 is highly upregulated in activated T lymphocytes and macrophages. TIPE2 expression is regulated by miR-21, and NF-κB regulated T-cell apoptosis via the miR-21-TIPE2 axis [33]. Recent studies have shown that miRNAs play a signifcant role in the pathogenesis of psoriasis by regulating immune responses at the translational level [34]. Additionally, genetic polymorphisms of some specifc miRNAs, such as miR-146a,  were found to be associated with psoriasis susceptibility [35]. Several studies indicated that various miRNAs are diferentially expressed in PBMCs from patients with psoriasis, which may be related to the decreased expression of TIPE2 [36][37][38]. Terefore, further confrmation is needed to determine the exact mechanism of TIPE2 downregulation in patients with psoriasis vulgaris.
As far as we know, the present case-control study is unique in assessing the expression of TIPE2 mRNA in the PBMCs of patients with psoriasis vulgaris and is the frst study to evaluate the possible correlation between TIPE2 and psoriasis. However, the present study had some limitations. First, the small number of psoriasis vulgaris samples included in the study, mainly from patients with moderate or severe psoriasis, might have afected the accuracy of the results. Second, the potential efects of drugs and other interventions on TIPE2 expression were not explored. Finally, a possible selection bias might have existed because the patient specimens were obtained from a single hospital. Terefore, our next study will investigate the potential infuence of antipsoriatic treatment on TIPE2 expression. Furthermore, TIPE2 silencing in cell lines or psoriatic mice should be investigated for the interplay between TIPE2 and psoriasis.

Conclusions
Our current study reported signifcantly reduced TIPE2 and elevated mRNA expression of other infammatory cytokines in the PBMCs of patients with psoriasis vulgaris. In addition, the expression levels of TIPE2, RELA, TNF-α, and IL-1β positively correlated with the PASI scores. Tese results indicate that TIPE2 may play an essential role in the pathogenesis of psoriasis vulgaris.

Data Availability
Te RT-qPCR data used to support the fndings of this study have been deposited in the 4TU. ResearchData repository (https://doi.org/10.4121/21836289.v1).

Ethical Approval
Te study was approved by the ethics committee of First Afliated Hospital of Xinxiang Medical University (No 2022045).

Consent
All participants signed the informed consent form.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Hua Hu and Xiuyu Fu contributed equally to this work. 8 Dermatologic Terapy