Interleukin-1β and Tumor Necrosis Factor-α Gene Polymorphisms in Systemic Sclerosis

Systemic sclerosis (SSc) is a generalized disorder of small arteries, microvessels, and connective tissue. It is a disease of unknown origin, with the highest incidence occurring between 45 to 55 years of age [1]; the frequency is three to eight times higher in females [2]. Several studies have demonstrated that the extent of skin involvement directly correlates with internal organ involvement and prognosis in SSc patients [3, 4]. Manifestations associated with SSc have been found to negatively impact the quality of life in affected individuals [5].

Long-term occupational exposure to environmental toxins is a common finding in SSc patients [6]. However, the effect of these environmental toxins on immune system of these genetically susceptible patients is unclear. Recent studies have raised the possibility that both genetic and environmental factors act synergistically at several stages of autoimmunity pathogenesis. These studies predict that individuals susceptible to spontaneous autoimmunity should be more susceptible following xenobiotic exposure by virtue of the presence of predisposing background genes [7]. Studies have shown that genetic predisposition plays an important role in susceptibility and the development of autoimmune diseases. This is likely due to functional polymorphisms within multiple genes, each of which, by modulating corresponding protein expression, influences disease susceptibility.

Cytokines may promote the deposition of collagen and fibrosis [8] and many studies have focused on the role of these mediators in SSc, enlisting alterations in their concentrations [9, 10] or in the balance between Th1 and Th2 cytokine levels [11]. Because cytokine production is regulated at the genetic level [12, 13], it has been hypothesized that single-nucleotide polymorphisms (SNPs) in or near cytokine genes may be relevant to the development of SSc.

Numerous studies examining patients with SSc from diverse ancestral backgrounds have identified SNPs in various cytokine genes. Among these, the IL-1 cytokine family has emerged as particularly significant in the pathogenesis of SSc. There is compelling evidence linking polymorphisms within the IL-1 family to the disease, as summarized by Xu et al. [14]. Specific gene variants in IL-1α [15], IL-1β and IL-2 [16,17], as well as IL-10 [18,19], have been observed across different populations. However, some associations reported in individual studies [19] have not been consistently replicated in others [17], highlighting the complexity and variability of these genetic links.

These and several other contradictions motivated us to investigate the commonly studied cytokine gene SNPs among our SSc patients, and compare our findings with those previously reported. In this study we evaluated the presence of 22 SNPs in 13 cytokine genes in SSc patients and attempted to associate the significant SNPs with SSc disease susceptibility in our population.

Cytokine production and release are key events in SSc pathogenesis as they are involved in T and B cell activation leading to inflammation, auto-antibodies production, microvascular damage and fibrosis [21]. The Th1/Th2/Th17/Treg balance is one of the hallmarks of SSc pathogenesis, as the Th2 and Th17 cytokines response leads to tissue fibrosis, whereas Th1 and Th17 cytokines promote inflammation in SSc patients.

IL-1α and IL-1β are proinflammatory cytokines involved in a number of autoimmune diseases. Patients who have SSc have increased circulating levels of IL-1α and IL-1β. Genetic associations with IL-1β have been investigated in patients with SSc and significant associations of the IL-1β -31 C and IL-1β -511 T alleles have been found [17]. Our results provide evidence suggesting that the T alleles of IL-1β -511 and IL-1β +3962 are associated with SSc in our population. Polymorphism in the human IL-1β gene has been reported to influence cytokine expression [22]. IL-1β stimulates the production of prostaglandin E2, which is an important cofactor for the induction of T-helper lymphocyte activity towards Th2 direction. A shift towards the Th2 system has been indicated in SSc [11].

Changes in IL-1β expression levels may reflect the genetic variation in IL-1β gene. The findings on biological roles of IL-1β polymorphisms, however, have not been consistent across studies. TT genotype of IL-1β -511 has been associated with higher gastric mucosa IL-1β levels in Helicobacter pylori positive population [23]. On the other hand, subjects with CC genotype showed an increased release of IL-1β from mononuclear cells after stimulation with lipopolysaccharide [24]. Recent studies suggest that the functional role of IL-1β -511 may depend on IL-1β promoter region haplotypes including IL-1β -511 [25]. Although the findings are inconsistent, these previous studies suggest that IL-1β -511 could affect the expression levels of IL-1β. On the other hand, the influence of IL-1β +3962 on IL-1β expression levels has not been previously reported. Polymorphisms in IL-1β, particularly SNPs IL-1β +3962 and IL-1β -511, have been identified as risk factors for susceptibility, progression, and severity of periodontal disease across various populations [26,27]. Elevated IL-1β levels in gingival crevicular fluid, saliva, and serum of periodontitis patients further support these associations [27]. Additionally, existing literature links inflammation associated with SSc etiology to the development of oral conditions like periodontitis [28]. Together, these findings suggest that IL-1β gene polymorphisms may contribute to the development of SSc in our study population.

A significant association of TNF-α -308 G allele and TNF-α -238 A allele with SSc was observed in this study. TNF-α, a member of TNF-superfamily, is a potent pro-inflammatory cytokine which affects different aspects of immune response, cell growth, differentiation and activation [29]. Due to its broad spectrum of pro- inflammatory functions TNF-α has been implicated in the pathogenesis of many immune disorders including all connective tissue diseases [29]. Increased production of TNF-α by PBMCs as well as elevated serum concentrations of TNF-α have been demonstrated SSc patients [30]. The enhanced production of TNF-α by PBMCs of SSc patients is associated with increased synthesis of TNF-α mRNA indicating increased expression of the TNF-α gene in SSc patients [31]. Moreover, elevated concentrations of TNF-α have been demonstrated in bronchoalveolar lavage fluid of SSc patients with interstitial lung disease [32]. Similarly, elevated serum TNF-α concentration in SSc patients were found in SSc patients with pulmonary fibrosis [33]. Recent studies have revealed inconsistent results regarding correlation of TNF-α polymorphisms with periodontitis susceptibility [34].

In our study, despite the patients and controls being of the same ethnic origin and from the same geographic region, we identified a strong association between the TNF-α -308 G allele and SSc. The functional significance and transcriptional impact of this allele remain a topic of debate, as some studies have found no direct link between TNF-α -308 polymorphisms and TNF-α production [35]. However, it is possible that an unidentified gene in linkage disequilibrium with the TNF-α -308 G allele may play a role in the increased susceptibility to SSc observed in individuals carrying this allele. In contrast, the TNF-α -238 A allele and GA genotype have also been previously associated with SSc [36]. Collectively, these findings suggest that TNF-α gene polymorphisms contribute to the pathogenesis of SSc in our study population.

The IL-10 gene promoter region contains several SNPs including –1082 G/A, –819 C/T and –592 C/A in the transcription factor-binding region. Alleles of all three polymorphisms are in linkage disequilibrium, giving rise to only three major allele combinations out of possible eight in Caucasian populations: the GCC haplotype is responsible for higher IL-10 secretion, whereas ACC and ATA haplotypes are associated with its lower production. Although we observed significant differences in the genotypes of the three IL-10 SNPs, no significant difference in haplotype (GCC/ATA/ACC) distribution between SSc patients and healthy individuals was observed. The G allele at -1082, and haplotypes containing this allele, have been associated with high IL-10 production, while the A allele and the ATA haplotype have been associated with low IL-10 production [37]. Our study does not show a possible correlation between IL-10 SNPs and its production in SSc patients. However, a significant association of IL-10 SNPs have recently been shown with chronic periodontitis [38].

The importance of TGF-β in SSc pathogenesis has been demonstrated well. TGF-β, its receptor, and downstream signaling molecules are expressed at increased levels in affected organs in SSc. TGF-β activates dermal fibroblasts leading to increased production of extracellular matrix. Given the importance of TGF-β in SSc, it has been hypothesized that polymorphisms in its gene may contribute to SSc susceptibility. However, there is a paucity of studies in this direction and the findings have been conflicting [39-41]. In this study we found no association between the SNP in TGF-β1 codon10 and SSc. However, significant differences were observed in TGF-β1 codon25 genotypes between patients and controls. Given the strong linkage disequilibrium among the SNPs in this gene, it is difficult, if not impossible, to assess which, if any, of these SNPs is truly responsible for the quantitative variation in TGF-β1 level. Possibly, particular alleles at these loci additively (or interactively) influence the quantitative (and possibly qualitative) expression of this cytokine.

The significant association between IL-1β (-511, +3962) and TNF-α (-308, -238) SNPs with SSc suggests a genetic predisposition to the disease, highlighting their potential as biomarkers for early diagnosis, risk assessment, and targeted therapies. These SNPs may contribute to SSc pathogenesis by dysregulating key inflammatory cytokines, promoting fibrosis and vascular damage. Additionally, they could help explain disease heterogeneity, offering insights into the severity of SSc and enabling better patient stratification. Given their role in other autoimmune conditions, these findings also provide broader implications for shared pathogenic mechanisms and therapeutic strategies, with the potential for inclusion in genetic screening panels for at-risk individuals.

Our study is limited by a small sample size and a restricted number of SNPs, as genetic susceptibility to SSc likely involves a broader combination of genes, along with environmental and epigenetic factors. Additionally, we did not adjust p-values for multiple testing, which complicates the determination of statistical significance and requires further investigation. While our findings suggest a potential association between IL-1β and TNF-α SNPs and SSc, larger studies are needed to confirm these results. The higher proportion of females in the SSc patient group compared to the control group reflects the well-known female predominance in systemic sclerosis [42], which is more common in women, especially in younger and middle-aged adults. While this sex distribution aligns with existing epidemiological data, the small sample size and sex imbalance may reduce the study’s statistical power and affect the generalizability of the results. Larger, more balanced studies are needed to confirm these findings and explore potential sex-specific genetic associations.

Future research should explore the functional roles of these SNPs through in vitro expression studies to evaluate their effects on cytokine production and immune cell activation. Employing genome-editing technologies like CRISPR-Cas9 could also provide insights into how these SNPs influence gene expression and cellular responses in relevant immune cells. Longitudinal cohort studies in diverse populations would further elucidate how these variants correlate with disease progression and treatment outcomes. These approaches would offer valuable insights into the mechanisms underlying SSc and help identify potential therapeutic targets, providing clearer directions for future studies in this field.

Our study found a significant association between IL-1β -511, +3962, and TNF-α -308, -238 SNPs and SSc in our population, consistent with previous research linking these SNPs to SSc susceptibility and progression. Larger, more balanced studies are needed to confirm these associations. Future research should focus on the functional roles of these SNPs through in vitro studies, genome editing, and longitudinal cohort studies to better understand their impact on disease progression and treatment outcomes.