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ORIGINAL ARTICLE
Year : 2022 | Volume
: 42
| Issue : 2 | Page : 137-140
Autophagy-related gene 16L1 value in chronic plaque psoriasis
Rehab M Naguib MD 1, Abd-El Aziz El-Rifaie1, Eman A.Z. Eissa1, Laila A. Rashed2
1 Department of Dermatology & Venereology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt
2 Department of Biochemistry, Cairo University, Cairo, Egypt
Correspondence Address:
Rehab M Naguib
Department of Dermatology, Beni-Suef, 19 Port Saed Street, Beni Suef, 62511
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejdv.ejdv_34_21
Background Psoriasis is a chronic hyperproliferative inflammatory disease, in which genetic and environmental factors have an important role, but the exact cause is yet unknown. Autophagy is a strictly regulated lysosomal degradation pathway that is crucial for maintaining intracellular homeostasis and normal development. Dysregulation of autophagy-related genes has been recognized to increase susceptibility to diseases, such as inflammation, cancer, and autoimmune disorders.
Aim Our study aimed to detect the expression of the autophagy-related gene 16L1 (ATG 16L) in psoriasis patients compared with normal controls to investigate the role of autophagy in the pathogenesis of this disease.
Patients and methods This case–control study included 30 psoriasis patients and 30 healthy controls. Punch skin biopsies of 4 mm were taken from psoriatic lesions and then from the controls and they were kept in a lysis solution for the stability of the studied parameters and were kept frozen at –80°C till analysis of ATG 16L using real-time PCR.
Results The level of the ATG 16L1 in the lesional skin of psoriasis was significantly increased compared with normal controls (P<0.001).
Limitation Limited number of patients were included in this study (30 patients).
Conclusion Autophagy process may play an important role in the pathogenesis of psoriasis disease.
Keywords: autophagy, autophagy-related gene 16L1, psoriasis
Psoriasis disease is a chronic inflammatory skin disorder, which affects ∼0.5–1% of children and 2–3% of the world population [1]. Psoriasis is considered to be a multifactorial disorder with many key components including environmental triggers, genetic susceptibility, with the skin barrier disruption and immune dysfunction [2].
Autophagy is a process that is detected in all cells at low levels under normal conditions, but many stimuli like hypoxia or starvation may lead to its upregulation. Cytoplasmic components are usually broken down into multiple basic components and then returned to be reused in the cytosol [3].
Autophagy is mediated by an organelle called the autophagosome. Autophagy is generally believed to be a nonselective degradation system. Besides mediating survival and homeostasis of the cells, autophagy is also important for antigen processing and presentation and in the secretion of proinflammatory cytokines like type I-interferon and tumor necrosis factor [3].
The autophagy-related gene 16L1 (ATG 16L1) molecule, which is encoded by the ATG 16L1 gene (2q37), is a key component of a large protein complex which is essential for autophagy; and polymorphisms in the ATG 16L1 gene contribute to the risk of psoriasis vulgaris [4].
The aim of our study was to detect the role of autophagy in psoriasis pathogenesis by the evaluation of ATG 16L1 expressions in tissue biopsies from the normal and psoriatic skin.
Patients and methodsThirty patients with chronic plaque psoriasis disease and 30 healthy controls were included in our study. Patients and healthy controls were recruited from individuals attending the outpatient clinic of Beni-Suef University Hospitals, Egypt in the period from June 2019 to March 2020.
Exclusion criteria included the use of any topical or systemic treatment for psoriasis disease in the last 3 months; patients with associated systemic diseases such as cardiovascular diseases and metabolic disorders including hypertension, diabetes, and dyslipidemia were excluded. Patients with other dermatological or autoimmune disorders were also excluded.
Patient information including age, sex, type of psoriasis, and the affected body surface area was collected by one dermatologist according to the rule of nines [5], and disease activity was determined by psoriasis area and severity index (PASI) score.
The aim of the study was explained to each patient, and an informed consent was taken from each patient. The protocol of the study conforms to ethical guidelines of the 1975 Declaration of Helsinki as reflected in the a priori approval by an Institutional Human Research Committee.
Estimation of autophagy-related gene 16L1 in tissues using quantitative reverse transcription PCR
RNA extraction
Total RNA has been isolated using the Qiagen tissue extraction kit (Qiagen, Chicago, IL, USA) according to the instructions of the manufacturer. Following this 30 mg of the human tissue sample was excised and placed directly into a suitably sized vessel for disruption and homogenization. The tissue was disrupted, lysed in lysis Buffer RLT, and the lysate was homogenized by a tissue homogenizer for 40 s. The lysate was centrifuged for 3 min at full speed, and the supernatant was removed carefully and transferred into a suitable microcentrifuge tube. One volume (350 μl) of 70% ethanol was added to the cleared lysate. Seven-hundred microliters of the sample was transferred to an RNeasy spin column placed in a 2 ml collection tube and centrifuged for 15 s at more than or equal to 8000 rpm. Five hundred microliters of buffer RPE was then added to an RNeasy spin column, and then centrifuged for 15 s at more than or equal to 8000 rpm to spin wash the column membrane. RNeasy spin column was placed in a new suitable 1.5 ml collection tube. A measure of 30–50 μl RNase-free water was added to the spin column membrane, then centrifuged for 1 min at more than or equal to 8000 rpm to elute RNA. The elute was carefully transferred to the new suitable Eppendorf tube and then stored at –80°C for further use. Purity (A260/A280 ratio) and concentration of RNA were then obtained using the spectrophotometry (dual wavelength; Beckman Spectrophotometer, Housten, Texas, USA).
cDNA synthesis
Total RNA (0.5–2 μg) was used for the cDNA conversion using the high-capacity cDNA reverse transcription kit (Fermentas, Housten, Texas, USA). One microliter of random primer was added to 10 μl of RNA, which had been denatured for 5 min at 65°C in the thermal cycler. The RNA primer mixture was cooled to 4°C. The cDNA master mix was prepared according to the kit instructions and was then added (for each sample). The last mixture was incubated in a programmed thermal cycler for 1 h at 42°C followed by inactivation of the enzymes at 95°C for 10 min, and then cooled at 4°C. RNA was then changed to cDNA. The converted cDNA was then stored at –20°C.
Real-time quantitative PCR using SYBR Green I
Real-time qPCR amplification were performed by using 10 μl amplification mixtures containing power SYBR Green PCR master mix (Applied Biosystem, Step One Plus, Elabscience Biotechnology Company Inc., Housten, Texas, USA), equivalent to 8 ng of reverse-transcribed RNA and 300 nm primers. The sequences of PCR primer pairs used for each gene are shown in [Table 1]. Reactions were then run on an ABI PRISM 7900 HT detection system. The PCR reactions consist of 95°C for 10 min (one cycle), 94°C for 15 s, and 60°C for 1 min (40 cycles). Data were analyzed with ABI Prism sequence detection system software and quantified using the v1.7 sequence detection software from PE Biosystems. Relative expression of the studied genes was then calculated using comparative threshold cycle method.
Statistical analysis
Data were coded and entered using the SPSS (Statistical Package for the Social Sciences Inc., Chicago, Illinois, USA), version 3.1. Data were summarized using mean, SD in the quantitative data, and using frequency (count) and relative frequency (percentage) for the categorical data. Suitable statistical tests were used (χ
2, one-way analysis of variance, one-sample t test, Pearson’s and Spearman’s correlation) whenever needed. P values equal to or less than 0.05 were considered statistically significant.
ResultsThe sex ratio and age were not substantially different for each variable among patients with psoriasis (20 men, 10 women; mean±SD age, 35.6±10.9) and healthy controls (22 men, eight women, mean±SD age 31.1±6.4). Clinical data of the participants are presented in [Table 2].
Table 2 Dermographic data, clinical characteristics of psoriasis patients, and controlsThe tissue ATG 16L1 expression.
The expression level of ATG 16L1 in psoriasis cases ranged from 2.2 to 9.10, with a mean value of 4.7±1.8, while in controls, it ranged from 0.97 to 1.2, with a mean value of 1±0.04, P value less than 0.001 ([Figure 1]).
Figure 1 Comparison between controls and psoriasis patients regarding ATG 16L1 gene expression in skin tissue biopsies. ATG 16L1 gene, autophagy-related gene 16L1.We found no relation between patient age, sex, disease duration, family history, PASI score with ATG 16L1 expression both in lesional and nonlesional tissue biopsies.
DiscussionPsoriasis, a chronic skin disease is known to be a more prevalent autoimmune disease in humans. The precise cause of psoriasis disease remains poorly understood. Psoriasis disease is thought to be due to a complex interplay between environment, genetics, immune dysfunction, and skin barrier disruption [2].
Psoriasis disease shows traits of autoimmune disease on top of an (auto)inflammatory background, with both mechanisms overlapping and even potentiating one another. Pathogenesis of psoriasis could be conceptualized into an initiation phase triggered by trauma (Koebner phenomenon), drugs, and infection followed by a maintenance phase characterized by a chronic clinical progression [6].
Psoriasis disease shows autoimmune-related pathomechanisms including abnormal multiple interactions among innate immunity, T cells, and keratinocytes. Moreover disturbances in innate and adaptive skin immune responses are responsible for initiation and sustainment of inflammation in psoriasis [7].
Immune cells in patients with psoriasis release more proinflammatory factors, leading to uncontrollable stimulation of both congenital and acquired immune systems, like nuclear factor-ΚB signaling pathways and also they cause differentiation of T helper (Th) cells toward Th1 and/or Th17 cells [8].
Autophagy is a regulated lysosomal degradation process, which is needed for maintaining the intracellular homeostasis and also normal development [3]. Autophagy is included in multiple innate and adaptive immune processes, like pathogen recognition and destruction, antigen processing for major histocompatibility complex presentation, regulation of lymphocyte development, and function and inflammation. Autophagy is needed for T-cell proliferation, differentiation, survival, and death. Autophagy is an important trafficking event in mediating T-cell response and regulating T-cell immunity [3].
Defects in ATGs and also recruitment of autophagy proteins are important for the autophagic dysfunction; defect in the regulation of ATGs has been detected to increase susceptibility to many disorders, such as autoimmune diseases like vitiligo, inflammation, and cancer [7]. There are studies that have shown that autophagy is related to multiple skin diseases [9].
There are also studies that have shown that autophagy dysregulation leads to production of inflammatory cytokines and cell proliferation in KCs [10].
The ATG 16L1 gene gives instructions for making protein, which is needed for the autophagy process. ATG 16L1 deficiency affects the autophagy machinery on the signaling pathways that regulate cytokine production and then lead to accumulation of the damaged proteins and organelles which are toxic, leading to tissue damage, cell death, and chronic inflammation [4].
All the above-mentioned facts prompted us to investigate the possible value of the autophagy process in psoriasis and in order to do so, we detected ATG 16L1 level in tissue.
We reported that the tissue level of ATG 16 in cases of psoriasis ranged from 2.2 to 9.10, with a mean value of 4.7±1.8, while in controls, it ranged from 0.97 to 1.2, with a mean value of 1±0.04. ATG 16L1 gene expression had no significant relation to patients’ age, sex, family history, disease duration, and PASI score.
This study was supported by Douroudis et al. [11], who reported that polymorphism in the ATG 16L1 gene is linked to susceptibility to psoriasis.
Also, Wenink et al. [12] reported that there was an increase in the expression of ATG 16L1 in DCs from patients with psoriatic arthritis.
ConclusionIn conclusion, autophagy may play an important role in the psoriasis pathogenesis, particularly through ATG 16L1; it can be used as a biomarker to evaluate its progression and effect of therapeutic interventions.
Study limitation
Limited number of psoriasis patients was included in our study (30 patients), so additional studies on a large number of cases is required to evaluate its exact role in the pathogenesis of psoriasis.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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