Evaluation of the role of YKL-40 in psoriasis and its connection to metabolic syndrome

Introduction

Psoriasis is a common dermatologic disorder with variable degrees of inflammatory severity. Skin lesions range from a few isolated erythematous scaly plaques to total body skin surface involvement [1]. For the past 30 years, the prevalence of psoriasis has risen across the globe, with considerable differences among ethnic groups. It ranged from 0.51% to 11.43% in adults and from 0% to 1.37% for children worldwide [2, 3]. In Egypt, a prevalence rate from 0.19 to 3% was found [4].
Despite being a chronic skin illness, psoriasis encompasses several harmful inflammatory events whose effects are not limited to the skin but are also responsible for the initiation of several coexisting disorders [5]. These include cardiovascular and gastrointestinal diseases, arthritis, mental health issues, and metabolic syndrome (MetS) [5, 6]. The association of psoriasis with those comorbid diseases has a significant impact on disease management and contributes to the reduction of life expectancy in psoriasis patients [7]. Metabolic syndrome is the most prevalent and significant comorbid illness associated with psoriasis, affecting 20% to 50% of patients [8, 9].
Metabolic syndrome is a cluster of abnormalities that puts the affected person at a higher risk of developing cardiovascular disease and type 2 diabetes mellitus [10]. These abnormalities include abdominal obesity, elevation of arterial blood pressure, hyperlipidemia, and glucose intolerance [11]. Both psoriasis and metabolic syndrome may arise as they share a common immunopathogenesis, such as persistent low-level inflammation related to several proinflammatory cytokines [9, 12].
YKL-40, commonly referred to as chitinase-3-like-1, is one member of the 18 glycosyl hydrolase gene group called chitinase-like proteins [13]. This glycoprotein is released by numerous cell types, such as activated neutrophils, macrophages, endothelial cells, blood vessel smooth muscle cells, and fibroblasts. Notably, various cytokines, like interleukin-6 (IL-6), IL-13, and IL-17, together with interferon-γ (IFN-γ) secreted by TH1 cells, control the YKL-40 expression in different cell types [14, 15]. The blood levels of YKL-40 were shown to be elevated in a wide range of human malignancies, autoimmune and chronic diseases [16].
Researchers were interested in examining the serum level of YKL-40 in psoriasis, psoriatic arthritis, and other autoimmune skin conditions, such as lupus erythematosus, Behçet disease, and rheumatoid arthritis, according to a review of the literature. Most participants showed elevated serum levels of YKL-40 in the autoimmune diseases studied. This was correlated with disease severity, indicating that YKL-40 could be a valuable diagnostic marker for inflammatory conditions [17]. Others reported reduced YKL-40 levels after treatment, especially in RA-treated patients with biological therapy as TNF-α inhibitors [18]. Furthermore, another study reported a reduction of its level in psoriasis patients following NB-UVB therapy. This suggests the possible use of this marker as a prognostic indicator [19].

Objective

The current investigation aims to evaluate the serum levels of the YKL-40 protein and its immunohistochemical (IHC) expression in the skin tissue of psoriatic patients. Then, we related these findings to the clinic-pathological features of psoriasis and assessed the parameters of MetS and carotid intimamedia thickness (CIMT).

Material and methods

The 70 participants included in this case-control study were split into two groups. In the patient group, 35 participants had chronic plaque psoriasis diagnosed according to the established clinical and histopathologic criteria of psoriasis. All patients had gone at least 1 month without receiving topical medications or 3 months of systemic medication before having a skin biopsy. In the other group, 35 control volunteers matched for age, gender, and site were included in this study. From May 2022 to July 2022, our patients and controls were randomly recruited from the Dermatology and Plastic Surgery Outpatient Clinics of Menoufia University Hospital, respectively. Each participant signed written informed consent form before the study started, which was authorized by the Committee on Human Rights in Research at our university (IPR number: 3/2021 DERM6).
The psoriatic patients under the study underwent the following:
1. Complete history taking was performed.
2. Clinical examination: general and dermatological examination (disease type and location) were conducted, followed by the application of Psoriasis Area Severity Index (PASI) score for clinical evaluation of the disease severity [20].
3. Anthropometric measures: to estimate body mass index (BMI), weight in kilograms with lightweight clothing and height in meters without shoes were measured. Body weight (kg) was divided by height in square meters (m²) and BMI (kg/m²) [21]. A non stretchable tape was used to measure the waist circumference (WC) at the midpoint between the lowest rib and the highest point of the iliac crest during normal expiration. The cut-off points of WC ≥ 90 cm in women and ≥ 95 cm in men were defined as abdominal obesity [22]. In a resting state, the average of two systolic and diastolic blood pressure measurements was calculated.
4. Radiological examination
Participants underwent carotid Doppler ultrasonography (US) using a 7.5 MHz linear array imaging probe. Participants were lying supine when the transducer was positioned in a longitudinal plane 1 cm proximal to the carotid bifurcation to maximize the lumen effectively. The distance between the lumen-intima and media-adventitia interfaces was used to calculate the CIMT of the far wall. Bilaterally, measurements were taken at 1 mm intervals from five adjacent spots, and the mean value was recorded. More than 1 mm in CIMT values was considered abnormal [23].
5. Laboratory investigations: In the same session, both blood sampling and skin biopsy were obtained from each participant at the Biochemistry and Pathology departments; each sample was processed independently and blindly as follows:
A. Blood samples:
Following a 12-hour nocturnal fast, each individual had 5 ml of venous blood drawn via sterile vein puncture and transferred into two tubes. Using a SPINREACT kit, Spain, 2 ml of blood was placed into a sodium fluoride tube for an enzymatic colorimetric assessment of blood glucose [24]. The remaining 3 ml of blood was drawn into a standard tube, allowed to clot at 37°C, and then centrifuged at 4000 rpm for 10 min. The clear supernatant serum was then extracted and stored at –80°C to estimate serum lipids and YKL-40. The enzymatic colorimetric test was conducted using a SPINREACT kit, Spain, to detect serum cholesterol and triglyceride (TG) [25, 26]. Serum HDL-c was assessed via the colorimetric method, using a human kit, Germany [27]. The Friedewald equation was employed to calculate low-density lipoprotein cholesterol (LDL-C) from cholesterol concentration, high-density lipoprotein cholesterol (HDL-C), and TG [28]. Serum YKL-40 was measured by ELISA technique using the Human YKL40 ELISA kit (Catalog No. 201-12-2064) (Chongqing Sunred Co., Ltd., China).
In instances where three or more of the following five requirements are satisfied, metabolic syndrome is diagnosed: waist circumference over 90 cm in women or 95 cm in men, blood pressure over 140/90 mm Hg, fasting HDL-C level less than 40 mg/dl (men) or 50 mg/dl (women) and fasting blood sugar over 100 mg/dl, and triglycerides greater than 150 mg/dl [29].
A person diagnosed with dyslipidemia is defined as having raised total cholesterol (TC), LDL-C, triglycerides, or lipoprotein(a) levels > 90th percentile or having levels of apo A-1 or HDL-C < 10th percentile [30].
B. Skin biopsies
Using a 3 mm punch tool, two skin biopsies were taken from each patient: one from a psoriatic lesion and the other from a perilesional skin 15 cm away from any psoriatic lesions. Likewise, each control individual was subjected to a site-matched punch skin biopsy. Regular processing of all biopsies was done in the Pathology Department. Two sections of 4-micron thickness were cut from each prepared block, one of which was cut on positively charged slides to be subjected to immunohistochemical staining. The other was stained with hematoxylin and eosin to determine pathological alterations.
Histopathological assessment: under a light microscope, sections stained by H&E were examined to establish the psoriasis diagnosis and quantify changes in the dermis and epidermis.
Immunohistochemical (IHC) staining procedure: The streptavidin-biotin amplified system was utilized as an immunostaining technique. Tissue sections embedded in paraffin undergo deparaffinization using xylene, rehydrated in a graded ethanol series, and then treated with hydrogen peroxide (3%). Then, the slides were washed with phosphate-buffered saline (PBS), followed by heat-induced epitope retrieval in citrate buffer solution (pH 6) for 20 min. Following cooling, the slides were coated with a monoclonal anti-YKL-40 antibody for the entire night at room temperature (Catalogue No. A3166, Chongqing ABclonal Co., Ltd, China; 100 µl concentrated and diluted by PBS in a dilution 1 : 200). This was followed by the incubation with secondary antibody using the Universal Dakocytomation Labeled Streptavidin-Biotin-2 system, horseradish peroxidase (LSAB-2 system, HRP kit, Catalog No. k0679). The chromogenic substrate and counter stain utilized were DAB and Mayer’s hematoxylin, respectively. The rat liver and spleen served as positive tissue controls, while a blocking buffer was used instead of the primary antibody to create the negative control.
Interpretation of YKL-40 immunostaining: The YKL-40 immunostaining was independently evaluated as positive or negative in the dermis and epidermis. Cytoplasmic staining was necessary to determine positive YKL-40 expression in any number of cells [31]. The intensity scores were assessed as follows: +1 for mild intensity; +2 for moderate intensity; +3 for strong intensity; 0 for absent staining. Additionally, numerical data (%) were employed to assess the expression percentage. The H score system was then implemented according to [32], where the following formula was used to account for both the intensity and proportion of positivity: H score = (3% × of strong intensity) + (2% × of moderate intensity) + (1% × of mild intensity). The maximum score was 3 × 100 = 300.

Statistical analysis

Using a personal computer running the “Statistical Package for the Social Sciences” (SPSS) version 22 application, data were gathered, tabulated, and statistically analyzed (SPSS, Inc.).

Results

Clinical and histopathologic data of the studied patients are presented in table 1.
Immunohistochemical expression of chitinase-like-protein YKL-40 in cases and controls All lesional and perilesional biopsies, in addition to controls, showed YKL-40 positivity. All layers of the lesional, perilesional epidermis (sparing granular layer), and dermal structures showed a cytoplasmic YKL-40 staining pattern. However, nuclear and cytoplasmic staining was observed in some cases. In controls, it appeared localized to the basal cell layer with cytoplasmic staining.
Comparison of chitinase-like-protein YKL-40 immunohistochemical expression between psoriatic cases and controls (table 2, fig. 1)
The percentage of YKL-40 expression and its H score were significantly higher in the lesional epidermis compared to controls (p < 0.001). Concurrently, adnexal expression also appeared significantly higher in cases than in controls, evidenced by increased YKL-40 percentage of expression and H score (p < 0.001).
Moreover, the expression percentages and H scores of YKL-40 in both epidermis and adnexa were significantly higher in perilesional cases than in controls (p = 0.001).
The mean H score of YKL-40 in the epidermis was significantly higher in lesional than perilesional specimens with mean ± SD of 180.6 ±48.6 vs. 172.5 ±48.2, respectively (p = 0.001).
Relation between H score of YKL-40 immunohistochemical expression in the epidermis and demographic, clinical, and histopathologic data together with data for metabolic syndrome and CIMT of the studied cases (figs. 2–4)
Psoriatic biopsies with decreased granular layer showed significantly lower H scores of YKL-40 expression in the epidermis than those with absent granular layer (p = 0.006). In contrast, biopsies with moderate dermal vascularity and inflammation showed higher H score values of YKL-40 expression in the epidermis than those with mild vascularity and inflammation (p = 0.001).
Relation between H score of YKL-40 immunohistochemical expression in fibroblast, blood vessels, and inflammatory cells with demographic, clinical, and histopathologic data together with data for metabolic syndrome and CIMT of the studied cases (figs. 2, 3, 5, 6) Cases diagnosed with dyslipidemia and metabolic syndrome showed significantly higher mean H score values of YKL-40 in fibroblast and blood vessels with inflammatory cells (p < 0.05).
The mean H score value of YKL-40 expression in fibroblast and blood vessels was significantly higher in patients exhibiting moderate vascularity and moderate inflammation in the dermis (p = 0.001 for all).
Patients with absent epidermal granular layer and those with a moderate degree of dermal vascularity and moderate inflammation exhibited significantly higher mean H score values of YKL-L40 expression in dermal inflammatory cells (p = 0.018, 0.001, and 0.001, respectively).
Correlation between YKL-40 H scores in the lesional epidermis and dermal components with demographic with clinical and laboratory data of metabolic syndrome and CIMT of the studied cases (table 3)
A significant positive correlation was found between YKL-40 H scores in lesional blood vessels and fibroblasts with total serum cholesterol and triglyceride (p = 0.030, 0.003, respectively).
Significant positive correlations between YKL-40 H scores in lesional inflammatory cells in the dermis with total serum cholesterol and triglyceride (p = 0.001, 0.0040, respectively).
Correlation between H score of YKL-40 immunohistochemical expression in the lesional epidermis and its H score in lesional dermal components (table 4)
There were significant positive correlations between YKL-40 mean H score values in the epidermis with YKL-40 H scores in dermal blood vessels and fibroblasts, dermal inflammatory cells, and adnexa in cases (p = 0.001).
Comparison between cases and controls regarding serum YKL-40 level (table 5)
Serum YKL-40 level was significantly higher in psoriatic cases than controls with mean ± SD 39.3 ±2.91vs 29.2 ±6.20, respectively (p < 0.001).
Relation between serum YKL-40 level and demographic, clinical, and histopathologic data together with data for metabolic syndrome and CIMT of the studied cases There was no significant relation between serum YKL-40 and demographic, clinical, and histopathologic data and data for metabolic syndrome and CIMT of the studied cases (p > 0.05).
Correlation between YKL-40 serum level and its immunohistochemical expression in the skin tissue of the studied patients (table 4)
There were significant positive correlations between serum YKL-40 level and H scores in the lesional epidermis, lesional blood vessels, fibroblasts, and lesional inflammatory cells in the dermis (p = 0.036, 0.049, and 0.009, respectively).

Discussion

In the current study, YKL-40 immunostaining showed positive expression in the epidermis in all layers (sparing the granular cell layer) and dermal components of lesional and perilesional skin samples with predominant cytoplasmic staining with some nucleocytoplasmic staining. In control cases, YKL-40 was observed in the basal cell layer of the epidermis with only cytoplasmic staining. Our results coincide with previous research [33–35]. Crucially, nuclear and cytoplasmic staining was noticed in some of our cases, and this was only reported in one previous study applying YKL-40 on breast cancer tissue [36]. Qin et al. claimed that this nuclear localization is due to improved antigen preservation in their tissues or a more sensitive immunohistochemical approach [36].
Upregulation of YKL-40 protein expression was observed in lesional skin samples compared to perilesional biopsies in addition to controls identified by increased percentage or/and H score values of its expression. To our knowledge, no previously published research on the immunohistochemical expression of YKL-40 in psoriasis exists. However, increased YKL-40 expression was observed in patients with other inflammatory skin diseases, such as lichen planus [37], hidradenitis suppurativa [34], and skin malignancy [33, 38]. There is a prevalent perception that YKL-40 staining was slight or absent in cells with no or recognized low levels of metabolism or proliferation. Thus, a positive correlation was observed between metabolic activity, proliferation, or turnover rate and YKL-40 expression [39]. This means that increased expression of YKL-40 in our cases could be related to the nature of psoriasis, which is a skin disorder with persistent inflammation and proliferation.
In particular, the sustained inflammation seen in psoriasis produced several cytokines leading to uncontrolled rapid epidermal proliferation with marked alteration of keratinocyte differentiation evidenced by the absence of a granular layer of epidermis [40]. Furthermore, early psoriatic lesions showed mild epidermal hyperplasia with focal loss of granular layer and mild dermal inflammation compared to late lesions [41]. Given the observation that YKL-40 protein expression becomes elevated in tissues with rapid proliferative rate and inflammation [42, 43], which is accepted to be more in late cases with absent granular layer, we could explain the increased YKL-40 H scores in epidermis and fibroblasts, blood vessels, and inflammatory cells in these studied cases.
Similarly, H score values of YKL-40 in epidermal and dermal components (fibroblasts, blood vessels, and inflammatory cells) indicated pronounced elevation in the studied cases with increased dermal vascularity and inflammation. Primarily, studies declared that YKL-40 could be expressed by keratinocytes, endothelial, vascular smooth muscle, fibroblasts, lymphocytes, and macrophages [16, 44, 45]. Additionally, YKL-40 is considered a marker of inflammation and tissue repair and remodeling [46]. YKL-40 appeared to be secreted explicitly by Th2 and Th17 cells, and it suppressed eosinophils, macrophages, T cell receptor-mediated cell death, and apoptosis [47, 48]. IL-17 (secreted by Th17 cells), which has a prominent role in psoriasis pathogenesis, is known to regulate YKL-40 expression. IL-17 can drive epidermal hyperplasia and the proinflammatory loop seen in psoriasis through its action on keratinocytes, endothelial cells, and immune cells [49]. Moreover, YKL-40 has a role in boosting angiogenesis via upregulation of vascular endothelial growth factor (VEGF), encouraging the development of branching tubules by endothelial cells [50]. All the above-mentioned effects of YKL-40 point to its potential function in psoriasis.
Notably, people with psoriasis experienced chronic inflammation with the release of several proinflammatory cytokines, which represent a fundamental factor underpinning the pathophysiology of MetS [51, 52]. Among those cytokines are TNF-α and IL-6 (released by infiltrating inflammatory cells of psoriasis), which have been described to stimulate the secretion of YKL-40 [53]. Concurrently, YKL-40 helps macrophages change into fat-rich foam cells, causing the development of atherosclerotic plaques. YKL-40 might potentially affect the adipocytes in a lipolytic manner, raising the plasma levels of atherogenic lipids [54]. Moreover, besides its role in inflammation, YKL-40 secreted by endothelial and vascular smooth muscle cells in psoriatic skin is suspected to enhance angiogenesis and endothelial dysfunction, contributing to atherosclerosis and cardiovascular disease progression [55]. Owing to the previously mentioned information, we could explain the significant association of YKL-40 expression by inflammatory cells, fibroblasts, and blood vessels in our studied cases with dyslipidemia and MetS and the significant positive correlation of its expression with total cholesterol and triglyceride blood level.
Considerably, keratinocyte proliferation, immune cell infiltration, and enhanced dermal vascularity are the main histological signs of psoriatic skin [56]. Furthermore, there is an established relation between cell proliferation, inflammation, and YKL-40 expression [39]. This is suspected to be a reason for the observed positive correlation between YKL-40 H scores in epidermal keratinocytes and its scores in dermal components (inflammatory cells, fibroblasts, blood vessels, and adnexa) in our studied cases.
Regarding the serum YKL-40 level, several studies reported higher levels of YKL-40 in patients with psoriasis vulgaris than in the control group, which agreed with our study [15, 57, 58]. When YKL-40 serum level was correlated with different parameters of psoriasis studied in those researches, conflicting results were obtained. Some showed a positive association between psoriasis parameters and YKL-40 [55, 59], while others reported no association in line with our results [57, 60]. However, this issue requires a deeper and more thorough analysis in further studies.
Salomon et al. suggested that elevation in YKL-40 serum level in the hidradenitis suppurativa cases could result from its secretion by inflammatory cells in lesional skin being strongly expressed by those infiltrating cells in the tissues [34]. Furthermore, it was found that YKL-40 was released by chondrocytes and synovial cells, as well as inflammatory cells in individuals with psoriatic arthritis, who have much greater plasma concentrations of YKL-40 [61]. Accordingly, we could explain the positive correlation between YKL-40 serum level and its H score expression values in epidermal and dermal components observed in the studied lesional psoriatic samples owing to its elevated expression by these cells.
Ultimately, our study substantiated the idea that psoriasis is not just a skin illness but a systemic inflammatory condition demonstrated by the elevation of all MetS parameters in the examined psoriatic patients. Moreover, it is worth noting that this is the first study investigating immunohistochemical skin tissue expression of YKL-40 in psoriasis in addition to its serum level. YKL-40 appeared to have a promising role in psoriasis as its tissue expression was associated with the most important pathologic changes seen in psoriasis, including increased dermal vascularity and inflammation. Furthermore, its tissue expression was associated with elevated lipid profile in our psoriatic patients, pointing to its possible role in developing metabolic syndrome.
Study limitations include: limited sample size, absence of distinct psoriasis clinical forms, no follow-up after treatment.

Acknowledgments

The authors sincerely thank the patients who participated in our study, the research team, and the healthy participants.

Funding

No external funding.

Ethical approval

Each participant signed written informed consent form before the study started, which was authorized by the Committee on Human Rights in Research at our university (IPR number: 3/2021 DERM6).

Conflict of interest

The authors declare no conflict of interest.

References