|Year : 2021 | Volume
| Issue : 1 | Page : 310-320
Nucleocytoplasmic expression of autophagy-related protein 7 in psoriasis
Rehab M Samaka1, Mohammed A Basha2, Wafaa A Shehata2, Ahmed E Tahoun3
1 Department of Pathology, Faculty of Medicine, Menoufia University, Shebin Elkom, Egypt
2 Department of Dermatology, Andrology and STDs, Faculty of Medicine, Menoufia University, Shebin Elkom, Egypt
3 Department of Dermatology, Andrology and STDs, Ministry of Health, Tala Hospital, Menoufia, Egypt
|Date of Submission||12-May-2019|
|Date of Decision||28-Jun-2019|
|Date of Acceptance||14-Jul-2019|
|Date of Web Publication||27-Mar-2021|
Ahmed E Tahoun
Menoufia, Shebin Elkom
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate the role of autophagy-related protein 7 (Atg7) in psoriasis.
Despite psoriasis being a common skin disorder, the exact pathogenic mechanism of psoriasis is still not fully understood. Abnormal autophagy is now established to be associated with pathogenesis of some skin disorders. Atg7 plays a central role in the machinery of the autophagy.
Materials and methods
This prospective case-control study included 30 patients with chronic plaque psoriasis, either lesional or perilesional, and 20 age-matched and sex-matched apparently normal participants as a control group. All cases were immunohistochemically stained for Atg7 antibody.
There was a significant difference among the three studied groups regarding epidermal topographic distribution of Atg7 (P < 0.001). Nucleocytoplasmic topographic localization was dominant in psoriatic skin, either lesional or perilesional, in comparison with 15.8% of normal skin. Regarding dermal Atg7 expression, there were significant differences between the studied three groups regarding all parameters of immunohistochemically of Atg7 such as status (P = 0.006), topography, percentage, and intensity (P < 0.001 for all).
Nucleocytoplasmic expression of Atg7 in psoriatic skin could be incriminated in pathogenesis of psoriasis.
Keywords: autophagy-related protein 7, autophagy, immunohistochemistry, psoriasis, skin
|How to cite this article:|
Samaka RM, Basha MA, Shehata WA, Tahoun AE. Nucleocytoplasmic expression of autophagy-related protein 7 in psoriasis. Menoufia Med J 2021;34:310-20
|How to cite this URL:|
Samaka RM, Basha MA, Shehata WA, Tahoun AE. Nucleocytoplasmic expression of autophagy-related protein 7 in psoriasis. Menoufia Med J [serial online] 2021 [cited 2021 May 8];34:310-20. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/310/312006
| Introduction|| |
Psoriasis is a chronic, immune-mediated, inflammatory skin disease . Despite the growing knowledge on molecular and cellular bases involved in the pathogenesis of psoriasis that has resulted in the development of biological therapies , till now, there is no curative treatment for psoriasis .
The efficacy of immune-targeting treatments has supported the immune hypothesis based mainly on dendritic cell and T-cell pathogenic functions. However, psoriasis cannot be considered uniquely as a T-cell-mediated disease, and it is well established that keratinocytes have a crucial role in triggering the early pathogenic events, as well as in sustaining the chronic phase of the disease .
The skin is a nutrient-poor environment, which is regularly exposed to various environmental stressors, and therefore, requires recycling of its limited resources. To perform its protective function efficiently without alarming the immune system, it continuously gets rid of dead cells . This housekeeping function is performed by autophagy . Autophagy is a complicated cellular mechanism that maintains cellular and tissue homeostasis and integrity via degradation of senescent, defective subcellular organelles, infectious agents, and misfolded proteins .
Autophagy is a double-edged process. It might have a pathogenic or therapeutic role in autoimmune diseases, where inhibition of autophagy ameliorates diseases including systemic lupus, multiple sclerosis, and rheumatoid arthritis. However, in other cases, it seems to exacerbate diseases such as psoriasis, psoriatic arthritis, and inflammatory bowel disease .
Genetic screening of patients with psoriasis revealed several single-nucleotide polymorphisms associated with autophagy-related gene 16L1 (ATG16L1), though the functional role of ATG16L1 variants in skin biology is unclear. In pustular psoriasis cases, mutations in adaptor-related protein complex 1 subunit sigma 3 (AP1S3), a gene encoding an autophagosome trafficking protein, resulted in the disruption of autophagy in keratinocytes and drived them to produce proinflammatory cytokines . Furthermore, autophagy inhibition in-vivo showed aberrations in keratinocyte differentiation, resulting in dysregulation of autophagy in psoriatic epidermis .
Autophagy-related protein 7 (Atg7) is a key regulator for autophagosome formation and one of the most important autophagy proteins, based on which many animal models are designed and established to investigate the role of autophagy in regulating cell physiological processes . To the best of our knowledge, the immunohistochemical (IHC) expression of Atg7 in psoriatic skin has not been studied before. The aim of this study was to evaluate the role of Atg7 in psoriasis.
| Materials and methods|| |
This prospective case–control study was carried out on 50 patients, including 30 patients with chronic plaque psoriasis and 20 age-matched and sex-matched apparently normal participants as a control group. Patients were selected randomly from Dermatology Outpatient Clinic, Menoufia University Hospital, Egypt, in the period between March 2017 and March 2018. Control participants were selected from persons attending Plastic Surgery Department. Despite the growing knowledge of molecular mechanisms involved in pathogenesis of psoriasis has resulted in the development of biological therapies.
Exclusion criteria were as follows: (a) cases of other types of psoriasis (guttate, erythrodermic, pustular, etc.), (b) patients who had received any topical or systemic therapy for at least 4 months, (c) any dermatological diseases except chronic plaque psoriasis, (d) any associated inflammatory or autoimmune disease, and (e) refusal of patients.
For each patient, a skin biopsy was taken from lesional skin and another biopsy from perilesional skin. The biopsies from the lesional skin were taken within the lesion, 1 cm from the edge of the plaque border . The biopsies from the perilesional skin were taken 2 cm beyond the plaque border .
All biopsies were submitted to Pathology Department, Faculty of Medicine, Menoufia University, Egypt. They were fixed in 10% neutral buffered formalin, dehydrated in ascending grades of ethanol followed by immersion in xylene, and then impregnated in paraffin. Several 5 μm-thick sections from each block were taken. One slide was stained by hematoxylin and eosin for routine histopathological examination. Other sections were mounted on positive charged slides and stored at room temperature for IHC staining for Atg7 antibody.
Clinicopathological parameters of studied psoriasis group
The following clinicopathological data were collected, including age, sex; onset, course, and duration of disease; site of affection; presence of itching; palm and sole affection; scalp affection; joint affection; nail affection; kobenarization; family history of psoriasis; and past history of similar conditions. Physical examination was done, including general examination and dermatological examination.
The severity of the disease was assessed by Psoriasis Area and Severity Index (PASI) score.
Histopathological changes in plaque psoriasis were assessed, and different degrees for each parameter were assessed subjectively. Epidermal changes included acanthosis, psoriasiform hyperplasia, hyperkeratosis, parakeratosis, Munro's microabscess, spongiform pustule of Kogoj, granular cell layer status, suprapapillary thinning, spongiosis, degree of spongiosis, tips of rete ridges, and epidermal inflammatory exocytosis. Dermal changes included elongation of papillary dermis, papillary dermal edema, dilation of blood vessels, tortuous blood vessels, inflammatory infiltrates, type of inflammatory infiltrates, and distribution of inflammatory infiltrates.
IHC staining was performed using rabbit polyclonal IgG antibody to Atg7 [100 μg/ml (GTX32459) GeneTex Inc. (North America), Irvine, California, USA]. Concentration was 0.1 ml, concentrated and diluted with EDTA 1: 100.
IHC staining was performed using the universal Dako cytomation labelled streptavidin–biotin-2 Santa Clara, CA, united states system. The primary antibody was applied on the slides and incubated overnight at room temperature in humidity chamber. Finally, the detection of bound antibody was accomplished using a modified labeled avidin–biotin reagent for 20 min and then PBS wash. A 0.1% solution of diaminobenzidine was used for 5 min as a chromogen. Slides were counter-stained with Mayer's hematoxylin for 5–10 min. Negative control slides were prepared by omitting the primary antibodies from the staining procedure. Positive control was normal gastric mucosa .
Expression of Atg7 in both epidermis and dermis was assessed in all studied groups: positivity, topographic pattern of expressions (nucleocytoplasmic, nuclear or cytoplasmic), and localization of Atg7 were assessed. Percentage of positive cells and intensity of staining were assessed.
It was conducted using statistical package for the social sciences (SPSS) program for Windows (version 20; SPSS Inc., Chicago, Illinois, USA). Fisher's exact and χ2-tests, least significant difference (post-hoc test), Mann–Whitney test, and Tamhan test were used. P value up to 0.05 is considered statistically significant and P value less than 0.001 is considered highly significant.
| Results|| |
Clinical data of the psoriasis group
Regarding the clinical data in this study, onset of disease was early in 19 (63.3%) cases and was late in 11 (36.7%) cases. Course of the disease was stationary in 20 (66.7%) cases and was progressive in 10 (33.3%) cases. Duration of the disease ranged from 0.33 to 18 years. Extremities were affected in seven (23.3%) cases, whereas both axial and extremities were affected in 23 (76.7%) cases. Scalp was affected in 16 (53.3%) cases. Palms and soles were affected in two (6.7%) cases. Koebnerization was positive in nine (30%) cases and was absent in 21 (70%) cases. Itching was complained by 23 (76.7%) cases and was not complained by seven (23.3%) cases. Joint affection was present in seven (23.3%) cases and was absent in 23 (76.6%) cases. Nail affection was present in 17 (56.7%) cases and was absent in 13 (43.3%) cases. Nine (30%) patients were smokers. Family history was positive in six (20%) cases and was negative in 24 (80%) cases. PASI score ranged from 1.8 to 35.5, with 10.19 ± 8.10 as a mean ± SD value. According to PASI score, 19 (63.3%) cases had mild psoriasis whereas 11 (36.7%) cases had moderate to severe psoriasis [Table 1].
Histopathological parameters of lesional and perilesional psoriatic skin
There were highly significant differences between lesional and perilesional skin regarding presence of acanthosis and its degree (P < 0.001 for both). There were also highly significant differences between lesional and perilesional skin regarding presence of psoriasiform hyperplasia and its degree (P < 0.001 and 0.02, respectively). Regarding presence of hyperkeratosis and its degree, there were highly significant differences between lesional and perilesional skin (P < 0.001 and 0.002, respectively). There were highly significant differences between lesional and perilesional skin regarding presence of parakeratosis and its degree (P = 0.04 and < 0.001, respectively). There was a significant difference between lesional and perilesional skin regarding presence of Munro's microabcesses (P = 0.002). There was a highly significant difference between lesional and perilesional skin regarding presence of suprapapillary thinning and its degree (P < 0.001 for both). There was a highly significant difference between lesional and perilesional skin regarding presence of granular cell layer (P < 0.001). There were significant differences between lesional and perilesional skin regarding presence of spongiosis and its degree (P = 0.001 and 0.049, respectively). There was a highly significant difference between lesional and perilesional skin regarding morphology of tips of rete ridges (P < 0.001). There was a highly significant difference between lesional and perilesional skin regarding presence of exocytosis and its type (P < 0.001 and 0.02, respectively; [Table 2]).
|Table 2: Comparison between lesional and perilesional skin of the psoriasis group regarding epidermal histopathological parameters|
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Immunohistochemical expressions of autophagy-related protein 7 in control group, perilesional skin, and lesional skin
[Figure 1], [Figure 2], [Figure 3].
|Figure 1: Immunohistochemical autophagy-related protein 7 (Atg7) expression in control skin. (a) Atg7 cytoplasmic localization in epidermis. (b) High-power view showed diffuse epidermal Atg7 cytoplasmic staining. (c) Diffuse cytoplasmic Atg7 staining in a hair follicle and eccrine sweat gland ducts. (d) High-power view showed cytoplasmic Atg7 staining with occasional nucleocytoplasmic localization (Atg7 immunohistochemically ×40 for a, ×100 for b and c and ×400 for d).|
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|Figure 2: Immunohistochemical autophagy-related protein 7 (Atg7) expression in perilesional epidermal psoriatic skin. (a and b) Diffuse nucleocytoplasmic Atg7 staining. (c) Atg7 nucleocytoplasmic staining was demonstrated in eccrine sweat glands and in a hair follicle. (d) High-power view showed Atg7 nucleocytoplasmic staining in a hair follicle (Atg7 immunohistochemically ×200 for a, b and d and ×100 for c).|
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|Figure 3: Immunohistochemical autophagy-related protein 7 (Atg7) expression in lesional epidermal psoriatic skin. (a) Positive Atg7 nucleocytoplasmic staining demonstrated diffusely in basal and suprabasal layers of epidermis é vanishing intensity in superficial layers. (b) High-power view demonstrating Atg7 nucleocytoplasmic staining in lower parts of epidermis. (c) Nucleocytoplasmic Atg7 staining was demonstrated in eccrine sweat gland ducts and a hair follicle. (d) Nucleocytoplasmic Atg7 staining was appreciated in eccrine sweat gland ducts (Atg7 immunohistochemically ×40 for a, c and d and ×200 for b).|
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Comparison between lesional and perilesional skin of the psoriasis group and control group regarding epidermal and dermal autophagy-related protein 7 expression
There was a significant difference between the three studied groups regarding epidermal topographic distribution of Atg7 (P < 0.001). Nucleocytoplasmic topographic localization was dominant in psoriatic skin either lesional or perilesional in comparison with 15.8% of normal skin. Regarding dermal Atg7 expression, there were significant differences between the studied three groups regarding all parameters of IHC of Atg7; parameters included status (P = 0.006), topography, percentage, and intensity (P < 0.001 for all; [Table 3]).
|Table 3: Comparison between lesional and perilesional skin of the studied cases and control cases regarding epidermal and dermal autophagy-related protein 7 expression|
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Relationship between autophagy-related protein 7 expression percentage (epidermal and dermal) and histopathological parameters
There was no significant association between Atg7 expression percentage and histopathological parameters [Table 4].
|Table 4: Relationship between autophagy-related protein 7 expression percentage (epidermal and dermal) and histopathological parameters|
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Relationship between autophagy-related protein 7 expression percentage (epidermal and dermal) and clinical parameters
There was no significant association between Atg7 expression percentage and clinical parameters [Table 5].
|Table 5: Relationship between autophagy-related protein 7 expression percentage (epidermal and dermal) and clinical parameters|
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| Discussion|| |
Psoriasis is a chronic, immune-mediated, inflammatory skin disease . The precise pathogenesis of psoriasis is unknown. It can be explained by immunological dysregulation as well as abnormal keratinocyte proliferation/differentiation . This study was carried out on 30 patients with psoriasis and 20 apparently normal healthy participants as a control group. In this study, we aimed to assess the role of IHC expression of Atg7 in psoriasis.
The histopathological findings in this study showed features consistent with psoriasis, but there was disparity between findings of other studies; this can be explained on the basis of varying degrees of activity of the disease. It is clear that there is a wide spectrum of histologic changes recognizable in psoriatic plaques ,.
In this study, overexpression of Atg7 was noted in the psoriatic skin (lesional and perilesional) compared with expression in normal skin. Ogmundsdottir et al.  stated that overexpression of Atg7 was mostly ascribed to altered autophagy function. To the best of our knowledge and after thorough search in English literature, no previous data concerning Atg7 IHC expression in psoriatic skin were revealed. Scarcity of the published data regarding expression of autophagy antibodies in skin diseases drived us to discuss and explain the current findings depending on few available other autophagy markers' data.
Yin et al.  showed increased expression of other autophagy markers such as sequestosome 1 (SQSTM1), Atg16L1, Unc-51 like autophagy activating kinase, AP1S3, and Atg6 in psoriasis.
Increased epidermal expression of the autophagy-related protein, SQSTM1, has been observed in psoriatic skin by Lee et al. . In patients with psoriatic arthritis, increased expression of autophagy-related protein Atg16L1 suggested autophagy involvement in psoriatic arthritis pathogenesis .
Unc-51 like autophagy activating kinase was expressed in suprabasal and granular layers of nonlesional psoriatic epidermis, whereas in lesional psoriatic epidermis, it was highly expressed in the whole skin thickness, except in the parakeratotic layers .
Sil et al.  found that mutations in AP1S3, a gene encoding an autophagosome trafficking protein, resulted in dysfunction of autophagy in keratinocytes and drived them to produce proinflammatory cytokines.
In their comprehensive report, Akinduro et al.  studied autophagy markers during epidermal differentiation. They noted that Atg6 was mainly expressed in the proliferating basal layer of healthy epidermis and shifts to basal and parabasal layers of nonlesional psoriatic epidermis and suprabasal layers of lesional psoriatic skin. In contrast, microtubule-associated protein 1A/1B-light chain 3 (LC3), a key marker of autophagy, was found to be expressed in all layers of healthy epidermis. In psoriatic lesions, LC3 was absent from all layers of the epidermis.
The expression of Atg7 beside the absence of LC3 in psoriatic epidermis, might be owing to the fact that Atg7 is responsible for activation of LC3. In psoriatic skin, Atg7 is malfunctioning, so LC3 would not be activated, and hence it was absent in all layers of psoriatic epidermis. Moreover, an isoform of Atg7 called [Atg7 (2)], detected by Ogmundsdottir et al. , was found to be unable to carry out the best characterized function of the protein, namely, the lipidation and activation of LC3
In this study, nucleocytoplasmic localization was dominant in psoriatic skin either lesional or perilesional in contrast to cytoplasmic expression in normal skin. Luo et al.  have highlighted the importance of nuclear autophagy in physiological and pathological processes. Our findings could throw light on the role of nuclear autophagy in psoriasis pathogenesis. Nevertheless, the underlying molecular mechanisms remain to be explored.
Several mammalian autophagy proteins were detected in the nucleus, including LC3, Atg5, and Atg7 . Keratinocytes are proved to undergo nucleophagy, autophagic degradation of the nucleus, and lack of nucleophagy in differentiating psoriatic keratinocytes was reported. These findings were correlated with parakeratosis in psoriatic skin . To clarify how Atg7 affects keratinocytes proliferation, Lee et al.  demonstrated that Atg7 could bind to the tumor suppressor p53 and had a role in regulating p53-dependent cell cycle. Complexes containing Atg7 and p53 complex were detected in both the cytosol and the nucleus.
In this study, we could assume that nucleocytoplasmic localization in lesional psoriatic skin may be responsible for exaggerated keratinocytes proliferation. However, how Atg7 is delivered to the nucleus is not fully understood. It may be owing to that nuclear lamina has been degraded before Atg7 has access to the nucleus . Further studies about the role of nucleophagy in psoriasis are recommended.
| Conclusion|| |
Nucleocytoplasmic expression of Atg7 in psoriatic skin could be incriminated in pathogenesis of psoriasis. However, further studies are recommended to clarify the role of autophagy in psoriasis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Eichenfield LF, Paller AS, Tom WL, Sugarman J, Hebert AA, Friedlander SF, et al
. Pediatric psoriasis: evolving perspectives. Pediatr Dermatol 2018; 35
Vide J, Magina S. Moderate to severe psoriasis treatment challenges through the era of biological drugs. An Bras Dermatol 2017; 92
Yin H, Wu H, Chen Y, Zhang J, Zheng M, Chen G, et al
. The therapeutic and pathogenic role of autophagy in autoimmune diseases. Front Immunol 2018; 9
Albanesi C, Madonna S, Gisondi P, Girolomoni G. The interplay between keratinocytes and immune cells in the pathogenesis of psoriasis. Front Immunol 2018; 9
Sil P, Wong SW, Martinez J. More than skin deep: autophagy is vital for skin barrier function. Front Immunol 2018; 9
Kim H, Park SY, Moon S, Lee J, Kim S. Autophagy in human skin fibroblasts: impact of age. Int J Mol Sci 2018; 19
Akinduro O, Sully K, Patel A, Robinson D, Chikh A, McPhail G, et al
. Constitutive autophagy and nucleophagy during epidermal differentiation. J Invest Dermatol 2016; 136
Li L, Chen X, Gu H. The signaling involved in autophagy machinery in keratinocytes and therapeutic approaches for skin diseases. Oncotarget 2016; 7
Alshenawy HA, Hasby EA. Immunophenotyping of dendritic cells in lesional, perilesional and distant skin of chronic plaque psoriasis. Cell Immunol 2011; 269
Komine M, Karakawa M, Takekoshi T, Sakurai N, Minatani Y, Mitsui H, et al
. Early inflammatory changes in the 'perilesional skin' of psoriatic plaques: is there interaction between dendritic cells and keratinocytes ? J Invest Dermatol 2007; 127
Hewitt SM, Baskin DG, Frevert CW, Stahl WL, Rosa-Molinar E. Controls for immunohistochemistry: the histochemical society's standards of practice for validation of immunohistochemical assays. J Histochem Cytochem 2014; 62
Ogawa E, Sato Y, Minagawa A, Okuyama R. Pathogenesis of psoriasis and development of treatment. J Dermatol 2018; 45
Raghuveer C, Shivanand DR, Rajashekar N. A clinico-histopathological study of psoriasis. Int J Sci Study 2015; 3
Bai S, Srinivasan S. Histopathologic diagnostic parameters of psoriasis; a clinicopathological study. Int J Res Med Sci 2016; 4
Ogmundsdottir MH, Fock V, Sooman L, Pogenberg V, Dilshat R, Bindesbøll C, et al
. A short isoform of ATG7 fails to lipidate LC3/GABARAP. Sci Rep 2018; 8
Lee HM, Shin DM, Yuk JM, Shi G, Choi D, Lee S, et al
. Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1. J Immunol 2011; 186
Wenink MH, Santegoets KC, Butcher J, van Bon L, Lamers-Karnebeek FG, van den Berg WB, et al
. Impaired dendritic cell proinflammatory cytokine production in psoriatic arthritis. Arthritis Rheum 2011; 63
Luo T, Fu J, Xu A, Su B, Ren Y, Li N, et al
. PSMD10/gankyrin induces autophagy to promote tumor progression through cytoplasmic interaction with ATG7 and nuclear transactivation of ATG7 expression. Autophagy 2016; 12
Dou Z, Xu C, Donahue G, Shimi T, Pan JA, Zhu J, et al
. Autophagy mediates degradation of nuclear lamina. Nature 2015; 527
Lee IH, Kawai Y, Fergusson MM, Rovira II, Bishop AJ, Motoyama N, et al
. Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress. Science 2012; 336
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]