Psoriasis is an immune-mediated chronic relapsing disease that affects both the skin and joints in a genetically predisposed individual 1. It affects two age groups, one major peak at 20–30 years of age and a later smaller peak of onset at 50–60 years 2. Multifactorial components comprising genetic susceptibility 3, and environmental triggers together with immune dysfunction 4, may participate in the psoriasis etiology, even though psoriasis etiology remains unknown.
These triggers activate dendritic cells (DCs) that migrate to the skin, draining lymphatics and activating antigen-specific T cells, to differentiate into effector T cells. They then reach the skin, where they release proinflammatory cytokines, stimulating the proliferation of keratinocytes with cytokine interaction between keratinocytes and T-lymphocytes 5. Interleukin 36 gamma (IL-36γ), formerly known as IL-1F9, is a member of the IL-1 family (IL-1F9). This family comprises the agonistic cytokines that include IL-36α (=IL-1F6), IL-36β (=IL1F8), and IL-36γ (=IL-1F9) as well as the antagonistic cytokine IL-36Ra (=IL-1F5). These cytokines are involved in the regulation of innate as well as adaptive immune responses 6. Emerging evidence indicates that IL-36γ signaling is involved in the activation of innate and adaptive immune responses 7.
The aim of the current study was to shed light on the possible hypothesized role of IL-36γ in the etiopathogenesis of psoriasis through its immunohistochemical expression in psoriatic skin lesions in comparison with normal skin biopsies, in addition to studying its association with the clinical aspects of the disease.
Patients and methods
This case–control study included 40 patients having psoriasis vulgaris, in addition to 20 nonpsoriatic apparently healthy volunteers as a control group. Cases of psoriasis were selected from Dermatology Outpatient Clinic, Menoufia University Hospital, during the period spanning from April 2015 to March 2016, while the controls were selected from those attending the Plastic Surgery Department. A written consent form was obtained from every patient before the study initiation. The study protocol was approved by the Research Ethics Committee of Menoufia University. Patients having psoriasis vulgaris from both sexes were selected randomly. They were advised to stop any topical (2 weeks) or systemic (1 month) treatment for psoriasis before joining the study. Any participant having immune-inflammatory diseases and/or uncontrolled systemic infection was excluded from the study.
The studied individuals were subjected to full history taking and clinical general examination to identify any excluding factor. The dermatological examination was performed, including psoriasis area severity index (PASI) score. PASI below 7 was defined as mild, between 7 and 12 as moderate, and above 12 as severe disease 8.
Stable chronic plaque psoriasis is characterized by predominant scaling, whereas erythema is the dominant feature of unstable progressing lesions. The transition to a more extensive involvement, due to frequently unidentifiable triggering factors, is frequently marked by the onset of inflammatory phase with predominant erythema, and limited scaling associated with itching and rapidly progressing lesions 9.
Punch biopsies (3 mm) were taken under 2% lignocaine local anesthesia from the involved skin of psoriasis patients and from matched sites of controls. Specimens were fixed in 10% formalin solution and sent to the Pathology Department, Faculty of Medicine, Menoufia University, for tissue processing.
From each specimen, two sections of 4 µm thickness were cut on routine slides, one for hematoxylin and eosin (H&E) staining, to assess the pathological changes including hyperkeratosis, parakeratosis, acanthosis, suprapapillary thinning, microabcesses, capillary proliferation and dermal inflammatory infiltrate 10, and the other for immunostaining. Purified mouse monoclonal antibody (Cat. #ab156783; Park Cambridge, UK) raised against IL-36γ received as concentrated 0.1 ml was optimally diluted at 1 : 100 using PBS. Negative control slides were prepared, by omitting the primary antibody from the staining procedure. While tissue sections prepared from human colon adenocarcinoma were used as a positive control for IL-36γ. The procedure of immunohistochemical staining was carried out according to the received datasheet of the used antibodies.
Immunohistochemically, the IL-36γ expression is confirmed by the presence of any cytoplasmic and/or nuclear staining 11. In case of positively expressed cells, the percentage of positive cells was assessed at ×400 magnification field 12, and intensity of the stain was subjectively graded as mild, moderate, or strong. Histoscore was calculated in all studied sections (histoscore=1×% of mildly stained cells+2×% moderately stained cells+3×% of strongly stained cells) 13.
The pattern of IL-36γ distribution was categorized as either patchy (irregular distribution) or diffuse (uniform in distribution), and its cellular localization was assessed as either cytoplasmic and/or nuclear. Epidermal cells stained by IL-36γ were grouped into: basal or suprabasal groups. The number of IL-36γ positive layers was either one cell layer (low expression), two to three cell layers (marginal expression), or more than or equal to 4 cell layers (strong expression) 14. In addition, inflammatory cells in the dermis were assessed for expression of IL-36γ (positive or negative).
Data were collected, tabulated, and statistically analyzed using a personal computer with statistical package for the social science, version 15 program (SPSS Inc., Chicago, Illinois, USA). Qualitative data were expressed as number and percentage. Quantitative data were expressed as arithmetic mean, SD, percentage, and median. For comparing qualitative variables, we used χ2 test and Fisher’s exact test, to evaluate exact probabilities in a contingency table (usually 2×2) when the expected frequencies were small. The Mann–Whitney U test was used in comparing two nonparametric quantitative variables. Kruskal–Wallis test was used in comparing three or more nonparametric quantitative variables. P value less than or equal to 0.05 was considered statistically significant.
Demographic data of studied patients
The psoriasis patients’ ages ranged from 11 to 75 years, with a mean of 43.45±18.10 years. There were 25 (62.5%) male individuals and 15 (37.5%) female individuals. As for the control group, 65% (13/20) were male individuals and 35.0% (7/20) were female individuals. The mean age of the controls (n=20) was 35.05 years. Psoriasis patients and controls were age and sex matched (P=0.071, 0.85, respectively) (Fig. 1).
Clinicopathological data of psoriasis patients
The clinical criteria and H&E histopathological changes of studied psoriasis patients are demonstrated in Table 1.
Interleukin 36 gamma immunohistochemical expression
In controls, IL-36γ showed positive expression in 16 (80%) sections, all of which were of low expression, detected in the basal cell layer only and revealed cytoplasmic localization (100%) (Fig. 2a). While in psoriasis patients (Fig. 1b and c) IL-36γ immunoreactivity was expressed in 35 (87.5%) cases, all of which were in the superficial layers (100%), which was mainly of nuclear localization (28, 70%), and showed strong expression (more than or equal to four layers) in 24 (68.57%) sections (P<0.001 for all) (Table 2).
Interleukin 36 gamma-positive versus interleukin 36 gamma-negative psoriasis patients
Compared with IL-36γ-negative psoriasis patients (n=5), IL-36γ-positive psoriasis patients (n=35) were mainly male individuals (60 vs. 20%), of younger age (42.91±18.78 vs. 51.20±10.92) and having a positive family history of psoriasis (34.3 vs. 20%). In addition, these patients had an earlier age of psoriasis onset (26.86±15.33 vs. 38.00±19.61), longer disease duration (16.78±13.07 vs. 14.40±14.48) and higher PASI score mean value (11.80±2.65 vs. 10.58±2.70). Furthermore, IL-36γ-positive psoriasis exhibited severe (21, 60% vs. 1, 20%) and more progressive disease (25, 71.4% vs. 2, 40%). However, due to the small number of cases in the IL-36γ-negative group (n=5), the difference between both groups could not reach the level of significance except, for disease severity (P=0.01) (Table 3).
With regard to H&E histopathological parameters, IL-36γ-positive psoriasis patients (n=35) showed significant higher parakeratosis and acanthosis than IL-36γ-negative cases (n=5) (P=0.001, 0.05, respectively) (Table 4).
Clinical and histopathologic criteria of psoriasis patients with regard to number of interleukin 36 gamma-positive layers
The studied positive IL-36γ immunoreactive psoriasis patients (n=35) were divided according to number of IL-36γ positive layers into two groups: group A (n=24), in which more than or equal to four layers showed IL-36γ-positive reactivity (strong expression), and group B (n=11), in which IL-36γ positivity was observed in less than four layers (weak and moderate expression).
Compared with group B, patients in group A revealed significantly higher PASI score mean values (13.14±2.82 vs. 11.38±2.46) (P=0.043) (Table 5).
The first evidence for the potential role of the IL-36 family in inflammatory skin diseases emerged in 2007 when Blumberg and colleagues generated transgenic mice expressing IL-36α/IL-1F6 in basal keratinocytes. These mice developed a psoriasis-like skin phenotype; in addition, the authors found an increased expression of IL-36α in the human psoriatic skin 15. These findings were also supported by Johnston et al.16 who found an increased expression of all IL-36 cytokines (α, β, γ) and of IL-36Ra in human psoriasis skin lesions as well as in two psoriasis mouse models by PCR and immunohistochemistry technique. In 2013, He et al. 17 reported a significantly higher expression of IL-36α, IL-36β, and IL-36γ in the psoriasis group than in the control group. Moreover, they found a strong correlation among the IL-36 family, including IL-36γ expression in psoriasis and MAPK expression, which is a key cytokine in the pathogenesis of psoriasis.
The aforementioned results are confirmed by our findings with regard to IL-36γ tissue expression in psoriatic skin lesions. We selected IL-36γ to study, as IL-36γ, not IL-36α or IL-36β, was found to be a helpful histologic marker for psoriasis. In addition, it was defined as a cluster of IL-17/tumor necrosis factor (TNF) associated genes specifically expressed in psoriasis. Therefore, it might provide a future drug target due to its potential amplifying role in TNF-α and in the IL-17 pathway in psoriasis skin inflammation 14.
Moreover, we revealed that a strong expression of IL-36γ (more than or equal to four cell layers) was found only in IL-36γ-positive psoriatic sections (68.57%) versus none in the controls who showed only low expression (one layer) in all IL-36γ control slides. Furthermore, we observed this strong expression in nuclei (70%) of superficial epidermal layers of all IL-36γ-positive psoriatic lesions (100%), but the low expression in controls was of cytoplasmic localization in the basal cell layer (100%) (Figs 2 and 3).
In accordance with our result, D’erme et al. 14 reported strong IL-36γ expression (more than or equal to four layers) in psoriasis cases, whereas low expression (less than four layers) was observed in their other studied diseases (atopic dermatitis, lichen planus, and contact eczema) and controls (P<0.05). Likewise, Boutet et al.11 in their recent work, reported that IL-36γ is highly expressed in the involved psoriatic skin compared with their matched controls (P=0.001).
On the basis of several mouse models of skin inflammation, overexpression of IL-36 in mouse skin leads to a disease quite similar to human plaque psoriasis, and inhibition of IL-36 in human psoriatic skin ameliorates the inflammation 18. This psoriasis, like dermatitis in mice, is driven by IL-36-mediated DC keratinocyte crosstalk 19. Human keratinocytes derived from patients with psoriasis have been shown to express significantly higher levels of IL-36γ in response to IL-17 than those isolated from healthy donors. In addition, the IL-1 family induces T helper type 17 (Th17) cells to produce IL-17. These observations thereby indicate that the IL-17/IL-1 axis plays important roles in the pathogenesis of psoriasis 20.
Furthermore, all three IL-36 agonists including IL-36γ induce proinflammatory cytokines, chemokines, and costimulatory molecules, thus promoting neutrophil influx, DC activation, the polarization of Th1 and IL-17-producing T cells (α, β T cells and γ, δ T cells) and keratinocyte proliferation 21.
In the present study, IL-36γ immunoreactivity was associated with significantly higher parakeratosis and acanthosis, the result that was not mentioned before. Moreover, the strong expression of IL-36γ was significantly (P=0.043) associated with a severe form of psoriasis in our studied cases, indicated by higher PASI score mean values. This result was supported by that of D’erme et al. 14 who reported that IL-36γ was specifically expressed in psoriasis skin lesions and closely associated with disease activity. Moreover, they revealed that IL-36γ serum levels were closely associated with PASI score and decreased under treatment with anti-TNF-α drugs.
Furthermore, Boutet et al.11 observed not only significant positive correlation of IL-36γ expression and psoriasis activity but also its significant positive correlations with mRNA expression of many cytokines including IL-36β, Th17 cytokines (IL-17a, IL-22, IL-23, and CCL20), OSM (IL-6 family member), interferon-γ, IL-8, and antimicrobial proteins. All of which are involved in the pathogenesis of psoriasis. Indeed, IL-36γ released by stimulated or damaged keratinocytes can activate dermal DC, leading to the secretion of proinflammatory cytokines. In addition, TNF-α and IL-17a can greatly promote the stimulatory function of IL-36 on cultured keratinocytes 22.
However, against these results, Watanabe et al. 23 found no significant relation between IL-36γ serum level and PASI score in their study on 14 psoriatic patients. They thought this may be due to the large number of their studied patients being under treatment or due to the evaluation of IL-36γ serum level using different enzyme-linked immunosorbent assay kits. In addition, the small sample size in their study may explain this difference. Moreover, Yazici et al.24 claimed that the PASI score index is a statistical rather than a dynamic method of evaluation of the intensity of psoriasis, and it does not reflect the real activity of the disease process.
The small sample size of the studied cases together with confinement of studying IL-36γ only at the protein level and not the gene level contributed to the limitation in the present study. Future consideration of a larger scale study together with studying the efficacy of anti-IL-36γ as a targeted therapy in the treatment of psoriasis is highly recommended.
IL-36γ may have an active role in psoriasis etiopathogenesis. Moreover, IL-36γ may be used as a target for immunotherapy in psoriasis management programs later on.
Conflicts of interest
There are no conflicts of interest.
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