Response of Human Toll–Like Receptor 2 During the Infection of Leptospirosis

Abstract

Purpose: Since Toll-like receptor2 (TLR2) plays a key role in mediating the innate immune responses in mice models against leptospirosis, we aimed to determine the response of human TLR2 during the infection of pathogenic Leptospira spp. Methods & Materials: Total RNA was extracted from patients' RNA stabilized whole blood followed by a DNase treatment. P-1 (messenger RNA specific) and P-2 (amplify both DNA and mRNA) primers were designed to target the human TLR2 gene. The most suitable primer pair was selected for the gene expression analysis, comparing the expression of human TLR2 using the two primer pairs. Gene expression analysis was performed using P-1 and the GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used as the housekeeping gene to calculate the gene expression value using the ΔΔCq (quantification cycle) method. Results: 64 patients were confirmed for leptospirosis, among 100 of clinically suspected patients using a previously validated qPCR. Among the confirmed patients, 18 were selected with Cq value <37 and with ≥20 ng/μL of RNA quantity for the current study. Optimized annealing temperatures were 56°C and 58°C for P-1 and P-2 respectively.Primer selection results; P-1 amplified only 1 of 10 samples,while P-2 amplified the human TLR2 gene in 7 out of 10.The corresponding no reverse transcriptase of 7 samples were amplified, that implied the presence of genomic DNA. Accordingly, P-1 was used as the primer in the gene expression study, that 1 of 18 showed a 'dr' (ΔΔCq=0.01352) and the rest showed 'no change' of the TLR2 regulation (ΔΔCq=0.0000). The 'dr' of the TLR2 gene resulted with mean Cq of 29 and 38 respectively for GAPDH and TLR2. Of the 17 resulted with 'no change', 12 didn't amplify the TLR2, but did amplify GAPDH with mean Cq values of 22,23,24,24,24,24,27,28, 30,31, 33, 37. The rest of the 5 samples didn't show gene amplification. Conclusion: The reported up-regulation of TLR2 in mice models wasn't observed in this study population and confirmed the usage validity of exon-exon spanning primers in gene expression studies where ever possible. Findings of human innate immune cell receptor responses give insights to develop new therapeutics against leptospirosis.