AA is a disease with an immunological base in different studies. The exact pathophysiology is still unclear and has not been determined (Gautam et al. 2020). It has been suggested that AA is an autoimmune process mediated by T cells and triggered by different stimuli, either endogenous and/ or exogenous, with the interaction of different molecules like cytokines (Ito et al. 2020). The hair follicle has an immune-privileged nature that protects it from autoimmune attacks (Żeberkiewicz et al. 2020). Different mechanisms are responsible for this, as the low number of T cells in anagen hair bulb (Moseley et al. 1997), absent major histocompatibility complex (MHC I) expression (Ito 2013), melanocytes are MHC I negative (Moseley et al. 1997), very low NK cell content in the anagen hair follicle (Ito et al. 2008), and multiple immunosuppressant molecules produced by anagen hair bulb (Welker et al. 1997). When there is a breakage of this immune balance, there will be lymphocyte infiltration in the hair follicle that appears as a “swarm of bees” pattern (Sperling & Lupton 1995). The infiltration was markedly observed in the acute stage, with an accumulation of T helper and T cytotoxic cells. The role of T cell subtypes and their cytokines in AA is still questionable and not well documented (Gautam et al. 2020).
In this study, two cytokines (TNF-α and IL-4) produced by T cells were chosen to study their polymorphisms (TNF-α rs1799964 and IL-4 gene intron 3 VNTR) in patients with AA. None of the selected have statistical significance when patients with AA are compared with normal controls (p -value > 0.05). Moreover, we got the same results when the genotypes were compared with the patient's clinical and demographic data (p -value > 0.05).
The TNF-α gene is located on the short arm of chromosome 6 next to the HLA class II region, which is highly polymorphic and contains many linkages disequilibrium, making it a major challenge (Asghar et al. 2004). TNF-α expression is regulated by several single nucleotide polymorphisms (SNPs) in the promoter region, the most important of which are 238, 308, 863, 857, and 1031 (Ghavami et al. 2017). TNF- α − 1031 (T/C; rs1799964) is considered as a functional SNP affecting gene expression as it lies close to the binding sites of 2 important transcriptional factors-kB and octamer Binding Transcription Factor 1(OCT-1) (Skoog et al. 2006; Sandoval-Pinto et al. 2016). The minor allele frequency (1031 C) is 22% in the 1000 Genome Database, and some studies have found that the C allele has higher transcriptional activity than the T allele (Higuchi et al. 1998; Zhou et al. 2018). To our knowledge, this is the first time this polymorphism has been tested in AA. However, it has been investigated in many disorders, either alone or associated with other polymorphisms, with conflicting results. Acute coronary syndrome (Sandoval-Pinto et al. 2016), leprosy (Silva et al. 2015), malaria (Hananantachai et al. 2007), endometriosis (Asghar et al. 2004), Peptic Ulcer (Sugimoto et al. 2007), gastric cancer (Yang et al. 2009), multiple system atrophy (Zhou et al. 2018), Behcet’s disease (Ahmad et al. 2003) and Crohn’s disease (Negoro et al. 1999) were all investigated. Moreover, its role in other skin diseases has been reported. A study from India involved 733 patients showed the significant association of – 1031 with the other four promotor polymorphisms are significantly associated with generalized vitiligo (Laddha et al. 2012a). In Bullous pemphigoid, the minor C-allele might be protective (Tabatabaei-Panah et al. 2019). A study on Spanish patients showed that − 238 and − 1031 polymorphisms might be risk factors in moderate and sever forms of psoriasis (Gallo et al. 2012).
The role of the other TNF-α promotor gene polymorphisms in AA has been studied. Galbraith and Pandey studied the association of -238 and − 308 with AA and found no differences between patients and controls as regard − 238, while for − 308 there was a significant difference between patchy AA patients and controls (Galbraith & Pandey 1995). The same results for − 308 were obtained from a study on 59 Mexican patients (Cristina et al. 2012). A study on Turkish patients found an association of -238 with AA and individuals with the GA genotype have a higher risk for AA (NAMDAR & AKCILAR 2021). On the other hand, two studies from Egypt and one study from Iran found no association between − 308 polymorphism and AA (Moravvej et al. 2018).
The IL-4 gene is located on the long arm of the fifth chromosome 5 (q23-31). It is about 10 kb in size, with four exons and three introns. It lies in a cluster of genes related to other cytokines; most of these cytokines are related to TH-2 cells (IL-3, IL-5, IL-9, IL-13, and IL-15, granulocyte colony-stimulating factor) (Karakus et al. 2013). The third intron contains a VNTR polymorphism span of about 70bp. IL-4 VNTR may alter the splicing site of mRNA, which produces different variants (Elghoroury et al. 2018). There are two alleles for this polymorphism: one with a 70bp deletion and the other with a 70 bp insertion. The deletion allele is rare, has 2 repeats with 183 bp, and is assigned as R1. The insertion allele is the frequent one, has 3 repeats with 253 bp, and is assigned as R2 (Kuran et al. 2019). The R1 allele was suggested to increase the expression of IL-4 and the R2 allele has a protective role in some studies (Vandenbroeck et al. 1997). Moreover, the R2R2 genotype is associated with decreased IL-4 levels (Salimi et al. 2014).
The role of IL-4 VNTR has been evaluated in different disorders with or without an immunological basis with different results like prostate cancer (Bingöl et al. 2020), dysmenorrhea (Esen et al. 2020), end-stage renal disease (Ksiazek et al. 2019), type 2 diabetes mellitus and its complication (Ali et al. 2018), autoimmune hepatitis type 1 (Mansour et al. 2018), rheumatoid arthritis (Beh-Pajooh et al. 2017), immune thrombocytopenic purpura (Makhlouf & Abd Elhamid 2014) and multiple sclerosis (Karakus et al. 2013). In AA, there are very few studies regarding the role of this polymorphism. A case-control study from Turkey involving 116 patients with AA and 125 healthy controls showed that the R1 allele was statistically higher in patients with the R2R2 genotype when compared with the R1R2 + R1R1 genotype, a statistically significant difference was present. On the other hand, our study did not find any significant association when patients were compared with controls. This polymorphism was investigated in other dermatological disorders based on the immune system. In vitiligo, a study on the Gujarati Indian population found that both alleles and genotypes were significantly different when patients were compared with controls (Imran et al. 2012). In systemic lupus patients with cutaneous manifestation, the R1 allele was significantly different between patients and controls (Wu et al. 2003).
The study of cytokines is very essential as their role is very critical in the regulation of immune system response. Any change in cytokine levels either by increasing or decreasing, leads to immune system disturbance and may precipitate autoimmune response. Genetic polymorphisms are one of the most important mechanisms that affect cytokine levels by affecting gene expression. IL-4 is an immunomodulatory cytokine. It stimulates the differentiation of naïve CD4 T cells to TH-2 cells and enhances the differentiation and maturation of TH-2, and maintains its cytokines secretion. This leads to impairment of the balance between TH-1 and TH-2 cells, which precipitates an autoimmune response (Kidd 2003b). Moreover, it stimulates B cell immunoglobulin heavy chain class switch to IgE and increases IgE levels, which is the principal mediator of immediate hypersensitivity reactions (Imran et al. 2012). This may be an explanation for disease susceptibility and the association with atopic dermatitis (Katagiri et al. 2007). Also, it seems that TNF-α and IL-4 play in a harmony. TNF-α makes the surrounding micro-environment rich in IL-4 cytokine which facilitates IgE class switching (Altin et al. 2010). Moreover, it was found that the B cell-activating factor, which is considered one of the TNF-α family, increased in the sera of severe AA patients (Mackay & Leung 2006).
In conclusion, our results did not suggest any association between IL-4 gene intron 3 VNTR and TNF-α (rs1799964) gene polymorphisms and the occurrence of AA in Egyptian patients. The main limitations of this study, are that it was self-funded, which explains the small sample size plus the limited numbers of SNPs selected. Second, the disease pathology is complex and may involve environmental factors that were not evaluated. Third, all patients were from one canter, which means selection bias is unavoidable. Fourth, the previous studies regarding this issue were very few. Fifth, the limitation of the genotyping method.