This study investigated SNPs in genes encoding innate and adaptive immune responses thought to play key roles in the severity of AK from in vitro and in vivo animal and human studies. SNPs in five genes encoding biologically relevant molecules, CXCL8 (encodes IL-8, rs2227543 C/T, rs1126647 A/T, and rs2227307 T/G, all in 3'UTR gene region) and TLR-4 (rs4986790 A/G, rs4986791 C/T, missense coding gene region), IL-1β (rs16944, G/A, upstream regulatory region), IL-23R (rs11209026, G/A, missense coding gene region), and IL-22(rs1179251, C/G, intron gene region) were identified that were associated with SICs in AK after adjusting for covariates. SNPs of CXCL8 (encoding IL-8) and TLR-4 had two significantly associated SNPs, each in LD and exhibited the strongest association with SICs.
The
CXCL8 (encodes IL-8) SNPs were protective for SICs in this population. Carriers of the minor allele (T) were around 70% less likely to experience SICs than noncarriers in this study. In this same AK patient cohort, increased levels of IL-8 protein in tears were associated with more severe AK disease.
28 However, tear IL-8 levels with were not correlated with specific genotypes in patients in this study (
P = 0.324). Similarly, a study of sepsis susceptibility in Chinese men
29 found that the T allele for SNP rs1126647 was protective and increased levels of serum IL-8 were associated with sepsis in their population, but found no correlation between specific genotypes and the serum IL-8 protein levels. These findings indicate that this SNP may not be the causal SNP that increases the transcriptional capacity for IL-8. Other studies have suggested that rs4073 and rs2227306 may be causal SNPs.
30 Although significance associations of these SNPs were not found in this study, they are in LD with those identified as significant.
IL-8 is produced in the cornea by epithelial cells, keratocytes,
31 and macrophages
22 and has been shown to be upregulated in other ocular surface diseases, such as allergy
32 and dry eye.
33 IL-8 mobilizes and activates neutrophils, which is crucial in the early stage of corneal infection. However, activated neutrophils can also release metalloproteinases, which can degrade the extracellular matrix.
34 This is evidenced by an immunodeficient mouse model, which when infected with adenovirus vector encoding human IL‐8, results in neutrophil infiltration and corneal ulceration.
35 Thinning of the cornea and persistent epithelial defects, whereas not by definition part of SICs, are often present in severe cases of AK.
19 IL-8 is produced early in the inflammatory cycle, but unlike most other inflammatory cytokines, it can remain active up to weeks at the site of inflammation.
36,37 In contrast to the transitory effect of other proinflammatory cytokines that may last only a few hours, IL-8 may have a sustained influence
36,37 and therefore could be an important contributor to severe inflammatory outcomes in AK. In addition, IL-8 may be repeatedly produced by epithelial and inflammatory cells, leading to prolonged inflammation and collateral tissue damage.
Although IL-8 may have detrimental proinflammatory effects mediated through neutrophils, several animal studies have shown the powerful anti-acanthamoeba action of neutrophils in the early stages of disease. Neutrophils are chemotactic to
Acanthamoeba trophozoites and cyst lysates, and are capable of killing the organism in vitro through myeloperoxidase (MPO).
38 Upon injection of one million
Acanthamoeba castellani trophozoites into the anterior chamber of a mini pig model, AK was eliminated by a robust neutrophil response.
39 Intraocular infection has only been reported in a handful of human cases
8 indicating this neutrophil response in the anterior segment may be a factor in limiting the organism from infecting the posterior eye in humans.
In contrast to CXCL8, minor alleles of TLR-4 SNPs were associated with more severe AK in this study. Carriers of the minor allele were around 6.9 times more likely to experience SICs than noncarriers. The two significant TLR-4 SNPs are in almost complete LD. These SNPs are not common, however, the non-SIC minor allele frequencies are similar to population controls (minor allele frequency 0.06 from phase III 1000 Genomes) with increased frequency in SICs, reflecting a representative sample.
TLR-4 is a surface recognition receptor present on corneal and conjunctival cells. In the presence of
Acanthamoeba, in vivo and in vitro models both show activation of TLR-4 receptor.
40 In mouse models of AK, TLR-4 is also involved in initiating the cytokine complex.
40 It is likely that in patients with AK, decreased recognition of
Acanthamoeba allows a greater inoculum load into the cornea with dysregulation of the cytokine response, increasing disease severity. TLR-4 is primarily associated with a pro-inflammatory response, however, chronic activation of TLR-4 can lead to an anti-inflammatory response in some circumstances.
41 If this is the case in AK, patients with milder disease may be able to mount this anti-inflammatory response, whereas those with the SNP and more severe disease may not, leading to chronic hyperinflammation.
A study of patients with bacterial keratitis in India found higher carriage of
TLR-4 rs4986791 SNP compared with healthy controls.
18 However, another study did not find an association in rs4986790 (in LD with rs4986791) between CL patients with bacterial keratitis compared with healthy CL wearing controls in a Caucasian population.
14 The differences in results in these two studies are intriguing, but may be attributable to the rarity of the minor alleles. Interestingly, carriers of
TLR-4 SNPs have been shown to have a decreased response to endotoxins in several studies.
42,43 Of note, patients with
TLR-4 SNPs show a blunted airway response to inhalation of lipopolysaccharide (LPS).
42 The minor (T) allele of rs4986791 is associated with lower TLR-4 expression in blood plasma of a large German cohort, which is replicated in a Arab and Asian cohort of healthy individuals.
44
Three Th17-related genes were associated with SICs in this study,
IL-1β,
IL-23R, and
IL-22. IL-1β and IL-22 are proinflammatory cytokines and associated with the differentiation of CD4
+ T cells into effector Th17 cells.
For IL-22, the SNP (rs1179251), as for
CXCL8 (encodes IL-8) was protective. IL-22 tear fluid protein was also expressed more often in this cohort of patients with AK who experienced severe disease, as for IL-8.
28 However, we did not find an association between IL-22 tear protein levels and rs1179251 genotypes (
P = 0.07). To our knowledge, there are no studies that correlate IL-22 protein to rs1179251. Of note is that serum IL-22 protein is increased in idiopathic scleritis.
45 A study of IL-22 gene variations in autoimmune patients found the CC wild type genotype was associated with Graves’ ophthalmopathy, conferring protection by the minor allele, similar to our findings.
46 Serum levels of IL-22 are associated with the minor allele of rs2227484,
47 and stimulated peripheral blood mononuclear cells (PMBC) from patients carrying the minor allele of rs2227473 produce more IL-22.
48 However, both these SNPs are in the promotor region of the gene, whereas rs1179251 is in intron 4, rendering it difficult to draw comparisons.
In alignment with other cytokines in this study, the significant detection of SNP in
IL-1β (rs16944) is associated with a lower risk of SICs. This SNP minor allele is associated with increased gene expression in PBMCs.
49 However. IL-1β tear protein was not detectable in any of the tear samples from AK cases or nonaffected healthy controls in this current study population.
28 This SNP and others in
IL-1β have been associated with susceptibility to early onset periodontitis
50 and osteoarthritis.
51 In a previous study in a Caucasian population, an association was not found with
IL-1β SNPs between patients with CL associated bacterial keratitis and healthy CL wearing controls.
14 Further work is required to understand the role of
IL-1β in AK.
For
IL-23R rs11209026, carriage of the A allele was associated with a 6 times increased risk of SICs. The presence of the A allele increases expression of the soluble form of IL-23R mRNA (which then functions as a decoy receptor) and thus impairs the function of IL-23 in its ability to promote and maintain Th-17 cells.
52 This may mean that the Th-17 responses observed in an animal model of AK
23 are hampered in patients with this SNP.
There are several limitations of this study, in particular the sample size. Although this is a rare disease and we have a relatively large sample size per se, this limits power. In addition, the small sample size did not enable a more comprehensive analysis, such as genomewide association study (GWAS) or exome screening. Similarly, we have not made a correction for multiple testing of the 58 SNPs. All of the SNPs analyzed in this study had a priori hypothesis of association based on previous literature, but none reach significance under strict multiple testing criteria, despite the large effect sizes in many instances. Replication of these findings in similar, but independent, cohorts is critical and meta-analyses will likely be required to reach true statistical significance. Another limitation in this rare cohort was the self-report of ethnicity, which was completed poorly by participants (76/105, 72% identified ethnicity), however, 70/76, 92% identified as Caucasian. In a previous genetic study of bacterial keratitis in CL wearers at Moorfields Eye Hospital in 2012, 82% were Caucasian,
14 adding certainty that the cohort in this current study is predominantly Caucasian.
Nevertheless, the study author's clinical experience in this area meant a very well phenotyped cohort. In addition, a well-matched cohort in terms of sex and age between the SICs and non-SIC groups were recruited. Our results are further strengthened by an earlier published tear fluid protein study carried out on the same patient cohort.
28
In summary, our study has highlighted the importance of innate and Th17 immunity in patients with AK. A study of gene expression in late stage bacterial and fungal keratitis also found persistence of innate immune pathways.
53 Further work will be important to define cause and effect in tissue and animal models.
Furthermore, this study demonstrates that genetic screening of patients with AK for the presence of these SNPs is viable and could predict the risk of patients developing the severe inflammatory complications of the disease. The presence of these SNPs in an individual patient could assist clinicians managing AK by identifying patients susceptible to developing SICs in whom the prompt treatment of inflammation can be expected to improve outcomes.