Cytokine Profiles and the Relationship of Disease Severity in Patients with Psoriasis.

Background: Psoriasis is a chronic skin disease characterized by hyperproliferation of keratinocytes and increased inflammation. Previous studies have detected the levels of cytokines in the serum of patients with psoriasis, yet few multi-cytokine combination studies have been reported. Objective: The aim of the study was to compare the levels of cytokines in the serum between patients with psoriasis and healthy controls, elucidate which factors influence the psoriasis progression.
Methods: A total of 39 psoriasis patients and 30 healthy volunteers were enrolled. The venous blood was collected and the levels of 13 inflammatory cytokines were measured by human inflammation panel 1 kit. The severity of the disease was determined according to the psoriasis area and severity index (PASI) score.
Results: Compared with healthy controls, the levels of nine cytokines (IFN-γ, TNF-α, IL-1β, IL-6, IL-10, IL-12P40, IL-18, IL-17A and IL-23) were significantly increased, while the level of MCP-1 decreased in psoriatic patients. In addition, except for MCP-1, IL-10 and IL-12P40, these cytokine levels were positively correlated with the PASI score. Furthermore, there were higher serum lever of IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, IL-18 and IL-23 in active psoriasis than healthy controls and retrograde psoriasis. Conclusions: Increased serum levels of IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, IL-18 and IL-23 in psoriatic patients were associated with PASI and the stage of disease, which suggested that these cytokines play an important role in the pathogenesis of psoriasis. The detection of these cytokines can better observe the disease activity of psoriasis and optimize the treatment strategy. Copyright:

Introduction

According to the Global Psoriasis Report issued by the World Health Organization (WHO), the disease affects approximately 100 million people worldwide.[1] In the last three decades, the global incidence of psoriasis has been on the rise, bringing a severe economic and psychological burden to individuals and society.[2]

Psoriasis is a complex disease involving multiple risk factors including heredity, infection, immunity, etc.,It is currently believed that psoriasis can increase the incidence of risk of other metabolic diseases like insulin resistance,[3] hypertension,[4] myocardial infarction.[5] In addition, psoriasis can cause severe psychosocial disorders, which take adverse effects on the quality of life prolonging the course of disease.[67]

Psoriasis is characterized by scaly red plaques of epidermal hyperplasia, expansion of dermal blood vessels, and infiltration of inflammatory cells in the dermis.[8] Keratinocytes, immune cells, and endothelial cells interact with each other, leading to the occurrence of psoriasis. It is reported that psoriasis is mediated by T cells, which depends on the interaction between the innate and the adaptive immune system.[9] IL-23 secreted by dendritic cells (DCs) can promote the differentiation of Th17 and Th22 cells, which secrete cytokines such as TNF-α, IL-17, and IL-22. T cells that secrete high levels of IL-17 produce a self-amplified, feed-forward inflammatory response in keratinocytes, which aggravates keratinocyte proliferation and inflammatory cell infiltration.[910]

In addition, other cytokines are also related to psoriasis, such as IFN-γ, IL-6, IL-10, IL-12/23, IL-33 and so on. The serum level of IFN-γ in psoriasis detected by Roh et al.[11] was higher than that in the controls, but the result of Roussaki-Schulze et al.[12] was the opposite. Compared with the control volunteers, the serum levels of IL-12 of the patients with psoriasis were significantly increased,[13] still Kyriakou et al.[14] found that the median serum levels of IL-12/23p40 and IL-17 were not significantly different between psoriasis and healthy controls. At present, most of the researches are based on one cytokine, and there are few researches on the detection of multiple cytokines at the same time. The relationship between the levels of cytokines and the severity of psoriasis needs to be studied further.

In this study, flow cytometry was used to investigate the serum levels of 13 cytokines of patients with psoriasis. The research aimed to (a) determine whether the serum levels of 13 cytokines differs between the patients with psoriasis and healthy controls, (b) examine which cytokines are associated with psoriasis severity, and (c) assess whether other factors are associated with the serum levels of the cytokines.

Patients and Methods

Study design

Almost 39 patients with clinically confirmed psoriasis were enrolled from the Department of Dermatology and Venereal diseases from October 2019 to July 2020. The severity of the disease in patients with psoriasis was represented by the psoriasis area and severity index (PASI) score. The enrollees’ age, gender, family history, allergy history, body mass index (BMI), stages, and other clinical characteristics were recorded. Individuals with other skin diseases, abnormal blood coagulation, blood history, tumours, inflammatory bowel disease, acquired immune deficiency syndrome (AIDS) or other immunosuppressive diseases were excluded. Of these, 30 healthy volunteers were selected and their basic information were recorded during the same period. There was no significant difference in sex and age between the groups (P > 0.05). The study protocol was conformed to the ethical guidelines of the 1975 Helsinki Declaration. All participants signed the informed consent.

Samples and measures

About 3.5 mL of blood was collected from selected people in a sodium heparin anticoagulation tube, and then these samples were centrifuged at 4000 g for 10 min after coagulating for 30 min at room temperature. The supernatants were collected and saved at −80°C for testing.

A panel of 13 cytokines (IL-1β, IFN-α, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33) were measured using the LegednPlexTM Human Inflammation Panel 1 (13-plex) with V-bottom Plate kit (BioLegend, San Diego, United States) according to manufacturer's instructions.

Statistical analysis

The data were analysed using Statistical Package for Social Sciences (SPSS) soft, version 22.0. Chi-square test was used for sex, and the independent-sample T test was applied for age and BMI index. The Mann–Whitney test was used to analyse the difference in serum cytokine levels between psoriatic patients and healthy controls. The spearman's rank correlation test was used to evaluate the correlation between serum cytokines of psoriasis and PASI score. The differential levels of cytokines between stages were tested by the Kruskal Wallis test. The differences in different clinical characteristics of psoriasis were evaluated by the Mann–Whitney test of two groups and the Kruskal Wallis test of multiple samples. P < 0.05 was considered statistically significant.

Results

Patients and controls

A total of 39 psoriatic patients, 20 males (51.3%) and 19 females (48.7%) were enrolled in the study. The mean age and BMI of the recorded psoriatic patients were 28.01 ± 19.61 years and 22.82 ± 4.23. 5 (12.82%) patients with family history and 8 (20.51%) patients with allergies history were collected. The patients with psoriasis were divided into three groups according to the PASI score: mild 12 (30.77%), moderate 24 (61.54%), severe 3 (7.69%), and psoriatic patients were divided into three stages based on the Clinical Dermatology by Bian Zhao: 22 (56.41%) active stage, 6 (15.38%) stationary stage and 11 (28.21%) retrograde stage. The control volunteers comprised 30 subjects (16 male, 14 female, aged 40.20 ± 14.94 years, BMI 24.59 ± 3.69). There were no significant differences in gender (χ2 = 0.029, P = 0.866), age (t = −2.829, P = 0.380), and BMI (t = −1.828, P = 0.854) between the two groups [Table 1].

Table 1

Baseline demography and clinical characteristics of study groups

Project/Variable	Psoriasis(n=39)	Controls(n=30)	χ2/t值	P值
Sex (n, %)			0.029	0.866
Male	20 (51.28%)	16 (53.33%)
Female	19 (48.72%)	14 (46.67%)
Age, years (Mean±SD)	28.01±19.61	40.20±14.94	−2.829	0.380
Family history (n, %)
Yes	5 (12.82%)
No	34 (87.18%)
Allergy history (n, %)
Yes	8 (20.51%)
No	31 (79.49%)
BMI (Mean±SD)	22.82±4.23	24.59±3.69	−1.828	0.854
<18.5	6 (15.38%)	1 (3.33%)
18.5-24.9	21 (53.85%)	13 (43.33%)
≥25	12 (30.77%)	16 (53.33%)
PASI score (n, %)
Mild(0-3)	12 (30.77%)
Moderate (3-10)	24 (61.54%)
Severe (≥10)	3 (7.69%)
Stages (n, %)
Active	22 (56.41%)
Stationary	6 (15.38%)
Retrograde	11 (28.21%)

Mean values were compared between patients and controls using the independent-sample t test. Qualitative data were analysed by the Chi-square test. Data are presented as mean±SD and n (%). Abbreviations: BMI, body mass index; PASI, psoriasis area and severity index. SD, standard deviation. (*P<0.05, **P<0.01, ***P<0.001)

The serum levels of cytokines in patients and healthy controls

To indicate the differences in serum cytokine levels between patients with psoriasis and healthy controls, we detected the levels of serum cytokines in the two groups. We detected the levels of serum cytokines in the two groups. Compared with the healthy controls, the levels of IFN-γ (P = 0.005), IL-1β (P = 0.033), IL-6 (P = 0.002), IL-10 (P = 0.025), IL-12P40 (P = 0.019), IL-18 (P = 0.03) and IL-23 (P = 0.018) were significantly increased in patients with psoriasis [Figure 1a-g]. A more significant increase could be observed in the levels of TNF-α (P = 0.001), IL-17A (P < 0.001) in patients with psoriasis [Figure 1h, i]. However, the MCP-1 (P = 0.008) level decreased in the psoriasis patients [Figure 1j]. The levels of IFN-α, IL-8 and IL-33 were not analysed because few effective values were detected.

Figure 1

The serum l evels of IFN-γ (a), IL-1β (b), IL-6 (c), IL-10 (d), IL-12P40 (e), IL-18 (f), IL-23 (g), TNF-α (h), IL-17A (i) and MCP-1 (j) in psoriasis and controls. The Mann–Whitney U test was used in the comparisons. (*P < 0.05, **P < 0.01, ***P < 0.001)

Correlation between levels of cytokines in the serum and PASI score in patients

To further explore the correlation between cytokines and psoriasis progression, we conducted a correlation analysis between the levels of serum cytokines and PASI scores. The results showed that the levels of IFN-γ (P = 0.024, r = 0.360), TNF-α (P = 0.016, r = 0.382), IL-1β (P = 0.017, r = 0.379), IL-6 (P = 0.011, r = 0.401), IL-17A (P = 0.008, r = 0.420), IL-18 (P = 0.025, r = 0.359) and IL-23 (P = 0.001, r = 0.516) were significantly positively correlated with PASI scores [Figure 2a-h], while IL-10 (P = 0.051, r = 0.315), IL-12P40 (P = 0.056, r = 0.309), and MCP-1 (P = 0.355, r = 0.152) were not significantly correlated with the PASI scores [Figure 2i and j].

Figure 2

The correlations between serum levels of IFN-γ (a), TNF-α (b), IL-1β (c), IL-6 (d), IL-17A (e), IL-18 (f), IL-23 (g), IL-10 (h), IL-12 p40 (i), MCP-1 (j) and PASI were analysed by the Spearman rank correlation. (*P < 0.05, **P < 0.01, ***P < 0.001)

Cytokines levels in different stages of psoriasis

In this study, we observed the difference of cytokines that were positively correlated with the PASI score in different stages. Compared with healthy controls and retrograde stage, the levels of serum IL-1β, IFN-γ, TNF-α, IL-6, IL-17A, IL-18, and IL-23 in the active stage significantly increased [Figure 3].

Figure 3

Serum IL-1β (a), IFN-γ (b), TNF-α (c), IL-6 (d), IL-17A (e), IL-18 (f) and IL-23 (g) levels in different stages of psoriasis and controls. The Kruskal Wallis test was used to show differences of the comparisons. (*P < 0.05, **P < 0.01, ***P < 0.001)

Cytokine differences in other clinical characteristics of patients with psoriasis

To investigate whether other clinical characteristics of patients with psoriasis such as gender, age, family history, BMI influence the levels of serum cytokines, we selected these cytokines which were positively correlated with PASI scores for comparison and analysis with these factors [Table 2]. Our results indicated significant differences in the levels of IL-18 in different genders and ages. The serum levels of IL-18 in males were significantly higher than that in females (P = 0.015), and the level of IL-18 in age (18 ≤ age ≤ 40) was increased than that of minors (age <18) (P = 0.042). No significant differences were found between the groups in family history and allergies history.

Table 2

Cytokine differences in different clinical characteristics of patients with psoriasis

Project	IL-1β	INF-γ	TNF-α	IL-6	IL-17	IL-18	IL-23
Sex
Male	9.67 (1.08-19.82)	6.99 (4.87-11.55)	10.07 (5.71-19.86)	7.56 (5.48-10.81)	0.47 (0-1.28)	26.02 (16.73-32.61)	13.17 (4.74-26.96)
Female	5.87 (0-20.56)	5.67 (0.84-12.97)	6.88 (1.17-14.06)	6.17 (3.49-8.75)	/(0-1.32)	12.54 (7.63-27.67)	9.19 (0-27.86)
r	-0.597	-0.422	-1.714	-1.363	-1.3	-2.444	-1.213
P	0.551	0.673	0.087	0.173	0.194	0.015	0.225
Family history
No	6.96 (0-18.95)	6.36 (2.86-11.72)	8.2 (3.18-14.09)	6.81 (4.61-8.85)	0.08 (0-1.05)	18.75	12.08 (0.86-24.83)
Yes	25.01 (1.59-34.57)	10.11 (1.95-15.49)	20.86 (2.71-33.12)	11.07 (5.09-11.55)	1.75 (0-2.67)	13.45	34.32 (4.26-37.97)
r	-1.275	-0.504	-0.924	-1.029	-1.295	-0.21	-1.118
P	0.202	0.614	0.355	0.303	0.195	0.834	0.264
Allergy history
No	5.69 (0-18.69)	6.55 (1.13-12.18)	8.54 (3.13-16.12)	6.78 (3.84-8.75)	0.23 (0-1.32)	18.13	12.61 (0.88-27.63)
Yes	15.44 (5.03-36.25)	5.47 (3.72-13.96)	8.12 (5.78-13.2)	8.65 (5.36-12.01)	0.075 (0-1.15)	16.41	12.08 (2.73-33.36)
r	-1.477	-0.104	0	-1.165	-0.296	-0.591	-0.332
P	0.14	0.917	1	0.244	0.767	0.554	0.74
Age, years
>18	6.96 (0-15.54)	7.16 (1.06-13.01)	8.2 (1.39-20.75)	6.5 (3.57-9.91)	0.075 (0-0.93)	13.05 (7.98-20.24)	13.77 (0-25.69)
18-40	15.46 (0-23.9)	7.55 (4.41-12.69)	8.85 (3.17-16.56)	7.65 (5.54-10.55)	0.42 (0-1.45)	27.83	12.61 (1.43-35.26)
>40	5.3 (0.92-18.07)	5.42 (1.75-8.61)	7.19 (5.16-11.31)	6.08 (4.1-8.14)	0.17 (0-1.15)	22.87	11.71 (2.21-15.38)
r	1.143	1.277	0.337	0.814	0.443	6.343	0.381
P	0.565	0.528	0.845	0.666	0.801	0.042	0.827
BMI
<18.5	7.18 (0-20.53)	7.16 (1.06-11.18)	7.45 (0.81-21.19)	6.50 (3.44-19.75)	/(0-0.77)	10.43 (7.51-17.21)	20.60 (0-26.59)
18.5-2	10.61 (1.85-20.65)	7.55 (2.08-11.88)	8.85 (4.1-15.59)	6.78 (4.36-9.39)	0.34 (0-1.24)	19.36	12.40 (0.84-27.75)
>24.9	5.3 (0-18.21)	5.99 (3.59-13.14)	7.46 (3.75-14.16)	7.46 (5.24-9.67)	0.34 (0-1.40)	23.46	11.38 (4.06-25.96)
r	0.733	0.058	0.145	0.237	0.995	4.624	0.04
P	0.693	0.971	0.93	0.888	0.608	0.099	0.98

The Mann–Whitney test of two groups and the Kruskal Wallis test of multiple samples were evaluated the difference. Results are expressed as quartiles (*P<0.05, **P<0.01,***P<0.001)

Discussion

Related research showed that immune and inflammatory mechanisms play essential roles in psoriatic pathogenesis.[15] Especially, pathogenic T cell subsets such as Th1, Th17, Th22 and their characteristic cytokines including IFN-γ, IL-1β, IL-6, TNFα, IL-17, IL-23, chemokines and so on form the immune network of psoriasis through complex signal transduction.

Our findings indicated that the levels of serum cytokines TNF-α, IL-10 and IL-18 in psoriasis increased significantly compared with the healthy controls in this study, and this observation was in agreement with the results of Takahashi.[16] Mehta et al.'s[17] study on serum TNF-α level was also consistent with our results. TNF-α increases the expression of many mediators, such as IL-1, IL-6, IL-8, LTB4, PGE2 and adhesion molecules. Due to its multiple effects, TNF-α plays a vital role in the pathogenesis of psoriasis.[18] The role of TNF in the pathogenesis of psoriasis may be diverse. However, its crucial role is to promote the interaction between DCs and T cells, upregulate the composite of IL-23 in DCs, which accelerate Th17 differentiation.[19] IL-10 was related to the pathogenesis or development of autoimmune diseases. Borska et al.[13] indicated that the level of IL-10 in psoriatic patients was higher than that in the healthy controls, while Bieniek-Kobuszewska et al.[20] demonstrated that the serum level of IL-10 in psoriasis was lower than that in the healthy controls. Bai et al.[21] found that there was no significant difference in the level of IL-10 compared with the healthy controls. Although it shows different effects that sometimes seem to be contradictory. However, before further expounding the immunological environment that regulates its function, the ultimate role of this cytokine in disease needs to be further studied.[22] Bai et al.[21] found that the serum levels of IL-18 in psoriatic patients were increased significantly, which was consistent with our results. IL-18 may act synergistically with IL-23 to induce Th1 immune response without inhibiting the IL-23/IL-17 axis to exacerbate inflammation in psoriasis.[23]

In this study, we demonstrated that the serum levels of IL-17/23 in psoriatic patients were significantly increased, which is consistent with previous studies.[1124] The IL-23/Th17 immune axis was the main immune pathway of the pathogenesis of psoriasis.[25] IL-23 actives STAT3 through the JAK2 and TYK2 signalling pathways, which in turn leads to signal transduction in Th17 differentiated cells and produces a variety of cytokines, including IL-17A and IL- 17F, IL-22, IL-26, IFN-γ, CCL20 and TNF-α, the production of these cytokines exacerbates the chronic immune inflammatory process in patients with psoriasis.[2627] In psoriasis, IL-17 up-regulates the proliferation of keratinocytes and down-regulates their differentiation. In keratinocytes, IL-17 induces the production of chemokines (such as CXCL1 and CXCL8), attracting neutrophils to accumulate in the epidermis to form Munro microabscesses that are characteristic of psoriasis.[28] IL-23 (guselkumab) and IL-17 (secukinumab) inhibitors have been approved for use in moderate to severe plaque psoriasis, and have achieved good clinical efficacy.[29] IL-23 and IL-17 intracellular signalling inhibitors, such as TYK2 or RORγt, are currently in clinical development, and it is believed that they will benefit humans in the near future.[28]

In the study on the staging of psoriasis, our results showed that the serum levels of IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, IL-18 and IL-23 in the active stage were significantly higher than those in the degenerative stage and the healthy controls. A previous study found that the serum levels of TNF-α, IFN-γ, IL-6, and IL-18 in patients with active psoriasis were significantly higher than those in the controls, which were consistent with our results.[30] The serum level of IFN-γin the active and stable stages was significantly higher than those in the controls,[31] but we did not find the significant difference in the stable stages of IFN-γ level. Our results showed that the levels of TNF-α, IFN-γ, IL-6 and IL-18 in the active stage were higher than that in the degenerative stage, which may be due to the improvement of the disease in the degenerative stage and the decrease of pathogenic cytokines.

We detected that the serum level of IL-18 in male was significantly increased than that in female (P = 0.015), which imply that there are differences in the severity of psoriasis between male and female, but it requires a large number of samples to verify. Previous studies have shown that the median PASI score of women (5.4) in statistics was significantly lower than that of men (7.3) [P < 0.001], which is consistent in all age groups. Women were more inclined to accept local treatment, while men were more likely to receive systemic therapy.[32] People of any age can develop psoriasis, but two peaks can be observed: the first peak is between 20 and 30 years old, and the second peak is between 50 and 60 years old.[33] Our results showed that the serum IL-18 expression level of age (18 ≤ age ≤ 40) was higher than that of minors, which may be related to the peak incidence of psoriasis. The level of IL-18 was different in psoriasis patients of different age and gender, but its specific effect on psoriasis needs further research.

Conclusions

In summary, this study showed the cytokines levels (IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, IL-18 and IL-23) were increased in the serum of patients with psoriasis, and they were positively correlated with the PASI score. In addition, compared with the healthy controls and the retrograde stage, the serum levels of IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, IL-18, and IL-23 in the active stage were significantly increased. These cytokines may be related to the exacerbation of psoriasis, and the detection of these cytokines can better observe the disease activity of psoriasis and optimize the treatment plan.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

This research was supported by the National Natural Science Foundation of China (NSFC) (No. 32000006), the Science and Technology Research Project of Henan Province (No. 202102310270).

Conflicts of interest

There are no conflicts of interest.