Associations of TLR4 and IL-8 genes polymorphisms with age-related macular degeneration (AMD): a systematic review and meta-analysis

ABSTRACT Background The results of different studies have indicated the possible associations of TLR4 and IL-8 genes polymorphisms with Age-related Macular Degeneration (AMD). A meta-analysis study was designed to evaluate the possible associations of TLR4 (rs4986790/c.896A>G and rs4986791/ c.1196 C > T) and IL-8 (rs4073/c.251A>T and rs2227306/c.781 C > T) genes polymorphisms with AMD. Method A systematic literature search was carried out in PubMed, Embase, Web of Science, and Scopus databases to identify relevant publications. Pooled Odds Ratio (OR) with 95% Confidence Interval (CI) was used to evaluate the power of association. Results A total of 12 case-control studies with 4804 AMD patients and 4422 healthy controls were included in this meta-analysis. The analysis of genotypic and allelic models demonstrated significant associations between IL-8 c.781 C > T (CC vs. TT+TC: OR = 0.62 [0.48–0.81], P < .01; CC vs. TC: OR = 0.65 [0.48–0.89], P < .01; TT vs. CC: OR = 1.64 [1.04–2.57], P = .03; and C vs. T: OR = 0.71 [0.65–0.79], P < .01) and risk of AMD, which all of them passed Bonferroni correction for multiple testing (P-value≤0.01), except for TT vs. CC model. In addition, we found associations under the genotypic model of TLR4 c.896A>G (AA vs. AG+GG: OR = 0.73 [0.55–0.97], P = .03; and AA vs. AG: OR = 0.71 [0.53–0.95], P = .02) although after Bonferroni correction (P′-value<0.02) none of these associations remained significant. However, the data from this meta-analysis declined the associations of TLR4 c.1196 C > T and IL-8 c.251A>T polymorphisms with AMD. Conclusion The current meta-analysis study suggested that IL-8 c.781 C > T polymorphism is associated with susceptibility to AMD. Graphical abstract


Background
Age-related Macular Degeneration (AMD) is the leading cause of irreversible central vision loss in the geriatric population (1). Drusen as a primary visible clinical symptom of AMD are yellow sediments in the Bruch's membrane under the Retinal Pigment Epithelial (RPE) and photoreceptor cells (2). The advanced AMD can be classified clinically into dry (geographic atrophy) and wet (choroidal neovascularization) types. Although the exact cause of AMD is not entirely known, the results obtained from different studies have demonstrated that the environmental and genetic factors are possible risk factors for AMD. Non-genetic risk factors can be attributed to ageing, smoking, gender, race, body mass index, and high-fat diet (3). Based on the pathological features of AMD, the roles of abnormal inflammatory and immune responses have been demonstrated in the etiology of the disease (4).
Toll-Like Receptors (TLRs) play a key role in defending the immune system (5). TLR4 as a surface receptor for lipopolysaccharides (LPS) is highly expressed in lymphocytes, neutrophils, and monocytes (6). Chen et al. showed that the TLR4 signaling pathway is a possible mechanism involved in stimulating inflammatory and angiogenic factors in the RPE cells (7). Another study indicated that the mRNA level of TLR4 is increased in mice with retinal degeneration (8).
Interleukin-8 (IL-8) or C-X-C motif chemokine Ligand 8 (CXCL8) is one of the main inflammatory cytokines and a potent chemoattractant factor linked to the pathogenesis of AMD (9). Amyloid-beta as a drusen compound can induce inflammatory cytokine activation (such as IL-1β and IL-8) in the RPE cells (10). IL-8 is expressed by cells of the immune system, vascular endothelial, and the RPE cells (11). The investigations have suggested a possible impact of IL-8 level on inflammatory and angiogenic processes leading to the exudative AMD (12).
The meta-analysis method is a valuable statistical method used by combining multiple studies with the same subject to obtain a general result. This meta-analysis was performed based on eligible published literature to a more accurate description of the potential association between TLR4 (rs4986790/c.896A>G and rs4986791/c.1196 C > T) and IL-8 (rs4073/c.251A>T and rs2227306/c.781 C > T) genes polymorphisms and AMD.

Search strategy
A comprehensive literature search was conducted to identify all eligible studies regarding TLR4 and IL-8 genes polymorphisms and AMD risk in PubMed, Web of Science, Scopus databases for articles in English, and SID, Magiran, IranMedex, and IranDoc for articles in Persian up to April 2020 according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist and PICO approach (13). The following keywords were used: "Age-related macular degeneration OR AMD," "interleukin-8 OR IL-8 OR CXCL8 OR C-X-C motif chemokine ligand 8," and "Toll-like Receptor 4 OR TLR4." The flowchart of the article selection process is shown in Figure 1, which included a hierarchical approach based on title, abstract, and full-text reading. Moreover, all listed references of included studies and recent reviews were retrieved for any additional relevant studies.

Inclusion and exclusion criteria
Studies in this meta-analysis met the following inclusion criteria: (1) it was published by April 2020, (2) it was a case-control study or Genome-Wide Association Study (GWAS) that determined the distributions of the TLR4 (rs4986790 and rs4986791) and IL-8 (rs2227306 and rs4073) polymorphisms in AMD patients and controls, (3) detailed genotype frequency data could be acquired to calculate the Odds Ratios (ORs) and 95% Confidence Intervals (CIs), and (4) human studies. The exclusion criteria were: (1) abstract, case or series-report review, systematic review, animal studies, and publications in duplicate, (2) unrelated to each of AMD and these polymorphisms, and (3) insufficient data.

Data extraction
Briefly, first author's name, year of publication, ethnicity, mean age of participants, number of cases and controls, method for determination of genotype, studied polymorphisms, and Hardy-Weinberg Equilibrium (HWE) P-values were rigorously extracted by two investigators independently from the eligible studies. If there was a disagreement about data, the two investigators rechecked the original data of the included studies and had a discussion to reach a consensus; otherwise, the third investigator adjudicated the disagreements. Due to the lack of the mean age and HWE P-values for some studies, we were not able to analyze the data based on this information. This study was conducted in the Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences.

Statistical analyses
All statistical manipulations were performed with STATA version-15.0 software (STATA Corporation, College Station, Texas). The strength of the association between TLR4 and IL-8 polymorphisms and AMD was assessed by calculating pooled OR and 95% CI values in all genetic models; allele contrast, homozygous comparison, heterozygous comparison, dominant model, and recessive model for IL-8 polymorphisms and due to the small frequency of GG genotype at c.896A>G and TT genotype at c.1196 C > T, the correlation analysis was conducted by using allele contrast, heterozygous comparison, and dominant model. Z-test with P < .05 was used to authenticate the statistical significance of effect size. Sensitivity analysis was used to investigate the cause of dispersion. Then, outlier studies were removed and analysis was recomputed. In the case of a significant reduction in dispersion (I 2 index), this study was considered as a dispersion factor. Concerning the heterogeneity of the studies, Cochran's Q test (P-value [phet] < 0.10 was considered as statistically significant heterogeneity) and I 2 statistics (75 ≤ I 2 < 100 as extreme heterogeneity, 50 ≤ I 2 < 75 as high heterogeneity, 25 ≤ I 2 < 50 as moderate heterogeneity, and I 2 < 25 as no heterogeneity) were used to assess the degree of heterogeneity (14). The randomeffects model was used for this meta-analysis because it accounts for random variability both within and among studies. Publication bias was investigated by the Funnel plot and Egger's test. To avoid Type I Error, Bonferroni correction was conducted. For correction, 0.05 was divided by the numbers of comparisons. The output from the equation is α-value, which is a new threshold that must be reached for a single test to be considered statistically significant. In the present study, due to the multiple comparisons, five tests for IL-8 [c.251A>T and c.781 C > T] and three tests for TLR4 [c.896A>G and c.1196 C > T], the Bonferroni corrected P-value≤0.01 and P′-value<0.02, respectively, were considered statistically significant.

Literature search and study characteristics
The process of the search is shown in Figure 1. Relying on the search criteria and manual search of references cited in the relevant articles, 142 and 376 English and Persian language articles related to the TLR4 and IL-8 genes polymorphisms and AMD were initially identified, respectively. After eliminating the duplicated records and screening the titles, abstracts, and full texts, articles related to rs1927907, rs1927914, rs4986790, rs4986791 and A1062G for TLR4 and rs1126647, rs2227543, rs2227306 and rs4073 for IL-8 were extracted. However, polymorphisms for which there were no 3≤ eligible studies were removed. Finally, six studies for TLR4 (rs4986790 and rs4986791) and six studies for IL-8 (rs2227306 and rs4073) were included in this meta-analysis (wherein three studies were eliminated: one study due to non-availability of the data, even no responses could be gotten after having sought the relevant data via email contacts, one study due to the lack of the required number studies for analyzing of the polymorphism, and one study was included only patient's members).
The characteristics of the studies included in the meta-analysis are shown in Table 1. The genotypic frequencies of TLR4 and IL-8 polymorphisms in AMD cases and control are provided in Table  2. In total, our study included 4804 AMD patients and 4422 healthy controls. Of the 12 articles, studies including different subpopulations were considered as separate studies. Consequently, these groups were independently analyzed.

Association between TLR4 rs4986791/c.1196 C > T polymorphism and risk of AMD
In total, five studies (4,15,16,18,19), including 1714 AMD patients and 1074 controls were focused on assessing the impacts of TLR4 c.1196 C > T polymorphism and AMD susceptibility. The analysis of allelic and genotypic models showed no evidence of an association between this polymorphism and AMD (Table 3 & Figure 2).

Association between IL-8 rs4073/c.251A>T polymorphism and risk of AMD
Three studies (21,22,25) consisting of 865 AMD patients and 759 controls were enrolled in examining the association of IL-8 c.251A>T polymorphism with AMD susceptibility. The analysis of allelic and genotypic models failed to detect any association between this polymorphism and AMD. Also, the genotypic and allelic models analysis showed no evidence of association with wet and dry subtypes of AMD (Tables 3, 4 & Figure 2).

Tests for publication bias, sensitivity analyses, and heterogeneity
Publication bias was assessed by the Funnel plots, Metareg's test, and Egger test in the whole meta-analysis. A Funnel plot was not carried out for IL-8 c.251A>T because it is ineffective when the number of studies is limited. A significant asymmetry was found in AA vs. AG+GG model of TLR4 c.896A>G in the subtype analysis and C vs. T and CC vs. TC models of IL-8 c.781 C > T in the total and subtype analyses, respectively by the estimation of the Egger test P-values. Performing the sensitivity analysis by omitting the asymmetric study (21) (apparent asymmetry in the Funnel plots) and sequentially recalculating the overall effect in CC vs. TT +TC, CC vs. TC, and C vs. T models of IL-8 c.781 C > T in the total and subtype analyses led to altering the pooled OR and 95% CI (pre-sensitivity analysis data not shown in cases).
In this meta-analysis, significant statistical heterogeneities were found in most investigations. Thus, random-effect models were elected to synthesize the pooled ORs and 95% CIs for all genetic models. All of the control groups of studies were in HWE, except one study (22), which was non-assessed.

Discussion
AMD is an epidemic multi-factorial disorder triggered by a combination of genetic and environmental factors. While the contribution of genetics in susceptibility to the disease is estimated to be 45-70%, polymorphisms of genes encoding the immune  system and inflammation pathways play a major role in this heritability (26). In this meta-analysis, we aimed to determine the possible associations of the TLR4 (rs4986790/c.896A>G and rs4986791/c.1196 C > T) and IL-8 (rs2227306/c.781 C > T and rs4073/c.251A>T) genes polymorphisms with AMD risk.
IL-8 as an important chemotactic cytokine has a high level of production in the nAMD patient's PBMCs and RPE cells (36). A allele of c.251A>T and C allele of rs2227532 (c.845 T > C) polymorphisms in the promoter region of IL-8 gene cause higher expression of this gene (37,38). Most meta-analysis studies have demonstrated that IL-8 (c.251A>T) is associated with a higher risk of various cancer diseases such as gastric (39), prostate (40), breast (41), lung (42), and oral (43) cancers, while rs187238 (c.137 G > C) is associated with head and neck cancers (44).
In the current study, we utilized meta-analysis, in order to evaluate the association between the TLR4 (c.896A>G and c.1196 C > T) and IL-8 (c.781 C > T and c.251A>T) genes polymorphisms and the susceptibility to AMD. The relatively strong associations were observed in the comparisons of alleles and genotypes of IL-8 c.781 C > T polymorphism in both total and wet subtype of AMD (CC vs. TT+TC, CC vs. TC, and C vs. T). So that, individuals with CC genotype in IL-8 c.781 C > T have a 38% lower risk of developing AMD than the other genotypes. On the other hand, our meta-analysis disclosed no significant association in the comparisons of allele and genotypes models of TLR4 c.1196 C > T, TLR4 c.896A>G, and IL-8 c.251A>T in the total and subtypes of AMD analysis.
Luo et al. (45) reported the up-regulation of TLR4 in the retina of human glaucomatous eyes. The previous meta-analysis studies by AO et al. (46) and Shen et al. (47) demonstrated that the minor G allele of TLR4 c.896A>G polymorphism is associated with an increased risk of some inflammatory diseases, such as inflammatory bowel disease and Crohn's Disease. On the other hand, some functional investments have shown that Asp299Gly polymorphism causes deformation of the membrane structure, thereby the hyporesponsiveness of LPS (28) Ambreen's study (21) reported an elevated serum level for IL-8 in AMD patients versus the healthy control group. In another study by Jonas et al. (48), the finding suggested a direct relationship between the expanding of the choroidal neovascular membrane size in wet-AMD patients and the significant increase of the aqueous humor concentration of IL-8. Studies to evaluate the functional role of IL-8 polymorphisms have yielded different results. Hacking's study (49), which is in line with our finding, suggested that the T allele of c.781 C > T polymorphism contributes to promoting the binding of C/EBP β (as a transcription factor with a major role in the regulation of inflammation) within "−251A/+396 G/+781 T/+1238delA/+1633 T/+2767 T" haplotype box. Whereas, another investigation using the CRISPR/Cas9 system for IL-8 gene engineering by Benakanakere (50) demonstrated that carrying ATC/TTC haplotype in the SNPs of c.251A>T, rs2227307, and c.781 C > T shows an association with higher levels of IL-8 production in vitro compared with ATT/TTC haplotype. However, Ambreen's study (21) did not find any alteration in the serum level of IL-8 under the different genotypes of IL-8 c.781 C > T.
The probable reasons for the observed discrepancies may be owing to the mechanisms involved downstream or upstream of these genes and interallelic and intergenic interactions, which determine how the genes function in regulating inflammatory responses and thus the pathogenesis of AMD.
Several limitations in the present meta-analysis study should be brought up: 1) heterogeneity and publication bias, for instance, missing articles due to unpublished studies, possibly with negative results, and/or language bias due to writing in other languages (except English and Persian), 2) the small number of studies in some polymorphisms may lead to misleading results of the study, 3) further studies in other populations are essential for a powerful assessment, 4) joint effects of SNP-SNP or gene-environment factors, clinical diversities of AMD, as well as gender and age effects, were not investigated in this study due to the inaccessibility or limitations of data which could have significant impacts on the outcomes, 5) some genotype distributions of control groups do not follow the HWE.

Conclusions
In conclusion, the current meta-analysis study suggested that IL-8 c.781 C > T gene polymorphism is associated with susceptibility to AMD. Therefore, the presence of genotypes CC of IL-8 c.781 C > T polymorphism in individuals plays relatively a protective role against AMD. By a further understanding of the genetics and environmental factors involved in AMD pathogenesis, polymorphisms of the immune system can have potential implications for clinical trials, monitoring schedules, and preventive treatments implementation. Further large-scale and well-designed studies are required to validate this conclusion.

Study highlights
• The polymorphisms of genes encoding immune system and inflammation pathways play a major role in the heritability of AMD. • Toll-Like Receptors play a key role in defending the immune system. • TLR4 as a surface receptor for LPS is highly expressed in lymphocyte, neutrophil, and monocytes. • IL-8 or CXCL8 is one of the main inflammatory cytokine and a potent chemoattractant factor, which is linked to the pathogenesis of AMD. • The meta-analysis method is a valuable statistical method that is used by combining multiple studies with the same subject to obtain a general result.

Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.