Discussion
In this study, there was no statistically significant difference between CMI responding individuals in Egyptian HCWs with diverse HCV states with different TLR3.rs3775290 (c.1377C/T) genotypes. Also, there was no association between the results of the HCV-specific CMI response and the TLR3.rs3775290 (c.1377C/T) genotype among the 263 subjects tested. This agrees with another report [20]. TLR3’s function was investigated in four different viral models of infection in TLR3-/- mice, including lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), Murine Cytomegalovirus (MCMV), and reovirus. The researchers found that TLR3 is not always required for effective antiviral responses to be generated, as the absence of TLR3 did not alter either viral pathogenesis or the generation of adaptive antiviral responses to these viruses by the host. Remarkably, intracellular transduction of poly I: C signals begins TLR3-independent activation of an IFN response, therefore, restricting the role of TLR3 in the IFN pathway [21]. Other reports [22, 23] indicate that HCV may use TLR3 pathway to escape immune scrutiny through HCV NS3/4A protease-mediated cleavage of the TLR3 adaptor protein TRIF. Also, HCV NS3/4A impedes RIG-I, which is a key factor in TRIF-independent signalling [21]. In addition, it was suggested that several polymorphisms that alter TLR3 amino acids initiate resulting protein changes and that gene expression may be down-regulated while TLR3 activities required for proper signalling may be reduced. [24]. Lower TLR3 activity results in inability to identify the invading microorganisms and consequently insufficient immune responses. This increases the risk of infections and infectious diseases [25].
Our results provide strong indirect evidence that TLR9 could play a greater role than previously expected in HCV infection. We identified an association between the TLR9.rs5743836 polymorphism within the TLR9 gene and the natural history of HCV infection. In this study, there was a strong association between the results of the HCV-specific CMI response and the TLR9.rs5743836 (-1237T→C) genotype among the 265 subjects with valid CMI responses in total responders of individual HCWs (p=0.005). Also, there was a statistically significant difference between responding individuals in chronic HCV patients with distinct TLR9.rs5743836 genotypes (p = 0.044).
We show that the TLR9-1237T allele was significantly associated with HCV-specific CMI response. In this regard, the TT genotype is transcribed more effectively than the CC genotype as reported by others [26] who showed that the wild-type construct stimulates higher transcriptional activity compared to the CC allele. The data showed that individuals with the “favourable” TT allele would have HCV-specific CMI response when compared to those with the “unfavourable” CC allele. Also, several reports found that the mutant allele “T” imparts the immunity against the various pathogens. However, statistically inconsiderable, the mutant allele was declared to be linked with the depressed microbial load in Africans [27-29]. In addition, the mutant allele T manipulate immunity against establishing the infections, which is convincing that the TLR9 gene had bear the influence of genetic assortment to cope with the infections [30]. Functional analyses of the impact of TLR9 polymorphisms on basal promoter activity, on the other hand, revealed that the rs5743836 SNP causes higher gene expression than the wild type promoter. The findings of others [31] could explain the increased promoter activity of TLR9.rs573836. That study [31] showed that the variant C allele at rs5743836 establishes a potential NF-κB-binding site that increases the gene’s transcriptional activity . The presence of this extra putative NF-κB-binding site promotes transcription of TLR9 more effectively than the wild type TLR9.rs5743836 T sequence in response to different stimuli. This finding agrees with another report [32], where TLR9 promoter SNPs were associated with the natural course of HCV infection and exhibit higher transcriptional activities and imply the role of the DNA sensor TLR9 in natural immunity against HCV. Our data are analogous to other studies [1, 33], where the rs5743836 TT variant was reported to have a greater promoter activity than the CC genotype. That study suggested that this activity could lead to increased pro-inflammatory cytokine production during malaria infection, leading to successful malaria control and elimination [33].
There was no association between the results of the HCV-specific immune response and the TLR9.rs352140 (G2848A) genotype among the 258 Egyptian HCWs with valid CMI responses and TLR9.rs352140 genotyping. There have been no studies to date that indicate an association between TLR9.rs352140 (G2848A) and HCV infection. The region around 2848 is the major coding region of the TLR9 protein [34, 35] and a TLR9 2848 GA genotype polymorphism has been reported at the transcriptional level to reduce TLR9 expression [35, 36]. Down regulated TLR9 could reduce the functions of the innate immune response against HCV infection. Our results are related to another study that indicates that the TLR9 gene may play a role in cervical carcinogenesis but has a minor (or no) role in tumour progression where individuals with the favourable” GG allele were reported to be more frequent than the “unfavourable” AA allele [37]. In addition, a recent meta-analysis showed that there is no relationship between TLR9.rs352140 (G2848A) and cervical cancer vulnerability [38].  Our findings suggest that the TLR9.rs352140 (G2848A) genotype does not affect the natural course of HCV infection.
In conclusion, this study shows that TLR9.rs5743836 SNP; but not TLR3.rs3775290 or TLR9.rs352140 genotypes; could predict the outcome of HCV-specific CMI responses among genotype-4-infected Egyptians.
Acknowledgement : This study was supported by the Egyptian Science and Technology Development Fund (STDF) Contract no. 1664 to S.F. Abdelwahab.
Author contributions :
Conceptualization and design of experiments: SFA, SH, AO, ZZ, ER, and IW; Data collection, experimentation, analysis, and investigation: SH, SFA, MA, ZZ, IG, MS, WA; Funding acquisition and resources: SFA, ER, and IW; Writing-original draft (SH and SFA); Writing-review and editing (SFA, AO, MS, MH, WA, MA, ER, and IW). All authors approved the final version of the manuscript.
Compliance with Ethical Standards :
Conflict of Interest: None declared.
Data Sharing and Data Accessibility: All data pertinent to this article are included herein.
Research involving human participants and/or animals :
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments.
Informed Consent : Informed consent was obtained from all enrolled study participants.
Table (1): Relationship between TLR3.rs3775290 (c.1377C/T) and HCV-specific cell mediated immune (CMI) response among NLI HCWs in terms of CMI responders frequencies.