Discussion
In this study, there was no statistically significant difference between CMI responding subjects in Egyptian HCWs with different HCV states with different TLR3.rs3775290 (c.1377C/T) genotypes. Also, there was no relationship between the outcome 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]. The role of TLR3 was investigated in four different infectious viral models including lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), Murine cytomegalovirus (MCMV), and reovirus in TLR3-/- mice. The investigators found that TLR3 is not always required for the generation of effective antiviral responses, as the absence of TLR3 did not alter either viral pathogenesis or host’s generation of adaptive antiviral responses to those viruses. Interestingly, intracellular transduction of poly I:C initiates activation of an IFN response in a TLR3-independent manner, thus limiting the role of TLR3 in the IFN pathway [21]. Other reports [22, 23] indicate that HCV may use TLR3 pathway to evade immune surveillance via HCV NS3/4A protease-mediated cleavage of the TLR3 adaptor protein TRIF. Also, HCV NS3/4A interferes with RIG-I, a key factor in TRIF-independent signalling [21]. In addition, it was suggested that several polymorphisms that alter TLR3 amino acids initiate resultant changes in the protein and that they might downregulate the gene expression and lower the activities of TLR3 required for proper signalling [24]. Reduced activity of TLR3 results in failure to recognize invading microorganisms and insufficient immune responses, thus increasing the likelihood of infections and infectious diseases [25].
Our data provide strong indirect evidence that TLR9 might play a greater role in HCV infection than previously expected. We identified an association of the polymorphism TLR9.rs5743836 within the TLR9 gene with the natural course of HCV infection. In this study, there was a strong relationship between the outcome of the HCV-specific CMI response and the TLR9.rs5743836 (-1237T→C) genotype among the 265 subjects with valid CMI responses in responders of total subjects of HCWs (p =0.005). Also, there was a statistically significant difference between responding subjects in chronic HCV patients with different TLR9.rs5743836 (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 elicits higher transcriptional activity compared to the variant CC allele. Data showed that subjects 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]. In contrast, functional analyses of the impact of the TLR9 polymorphisms on basal promoter activity revealed that the rs5743836 SNP provoke higher gene expression compared with the wild type promoter. Increased promoter activity of TLR9.rs573836 might be explained by the findings of others [31], who showed that the variant C allele at rs5743836 creates a potential NF-κB-binding site that increased the transcriptional activity of the gene. The presence of this extra putative NF-κB-binding site promotes TLR9 transcription in response to various stimuli more effectively than the wild type TLR9.rs5743836T sequence. This finding agrees with another report [32], TLR9 promoter SNPs are associated with the natural course of HCV infection and show higher transcriptional activities and imply the DNA sensor TLR9 in natural immunity against the RNA virus, HCV. Our data are analogous to another study [1, 33], where the rs5743836 TT variant has been shown to have a higher promoter activity than the CC genotype. That study suggested that this activity could result in an increased pro-inflammatory cytokine production during malaria infection leading to successful control and elimination of malaria parasites [33].
There was no relationship between the outcome of the HCV-specific immune response and the TLR9.rs352140 (G2848A) genotype among the 258 Egyptian HCWs subjects with valid CMI responses and TLR9.rs352140 genotyping. To date, there have been no reports showing 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 polymorphism of the TLR9 2848 GA genotype has been reported to reduce TLR9 expression at the transcriptional level [35, 36]. Down regulated TLR9 could reduce the functions of the innate immune reactivity against HCV infection. Our data are analogous to another study which suggest that the TLR9 gene may play a role in cervical carcinogenesis but have a lesser (or no) role in tumour progression where subjects with the favourable” GG allele were reported to be more frequent than the “unfavourable” AA allele [37]. In addition, a recent meta-analysis demonstrated that there is no association between TLR9.rs352140 (G2848A) and cervical cancer susceptibility [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.