Birhan Alemnew^1,2,3,*, Tamirat Abebe¹, Soren Hoff⁴, Abraham Aseffa², Rawleigh Howe², Liya Wassie²

¹ Department of Microbiology, Immunology and Parasitology, College of Health Sciences, School of Medicine, Addis Ababa University; ²Armauer Hansen Research Institute, Addis Ababa, Ethiopia; ³Woldeya University, Woldia, Ethiopia; ⁴Statens Serum Institute, Copenhagen, Denmark.

*Corresponding Author: birhanalemnew12@gmail.com

Background: About one-third of the global population is considered to be latently infected with tuberculosis (TB) causing bacteria, Mycobacterium tuberculosis. Only 10% progress to active disease, while the majority contain the infection.  Latently infected individuals are considered as reservoirs for continuous new TB infections.

Objective: The aim of this study is to measure the mean fold change in mRNA gene expression of Toll-like receptors (TLRs) during latent TB infection as a diagnostic biomarker tool for detection of TB progression.

Methodology: Quantitative real-time PCR (qRT-PCR) was used to measure the expression of selected TLRs (TLR-1, TLR-2, TLR-4, TLR-6 and TLR-9) in a total of 64 cDNA samples, retrieved from AHRI biorepository and collected from 32 tuberculin skin test (TST) positive and 32 TST negative, using convenient sampling, apparently healthy school children and adolescents, aged between 11 and 20 years. Specific primers and florescent labelled probes were used to span exon-intron junctions to prevent amplification of genomic DNA. Human acidic ribosomal protein (HuPO) was used as an internal control. A comparative CT method was used to describe fold change in the relative expression of TLR genes. Data were analysed using Graph-Pad Prism 7.01 for Windows and a p-value less than 0.05 was considered statistically significant. This study was approved by the AHRI/ALERT Ethics Review Committee (AAERC), AAU-CHS IRB and the National Research Ethics Review Committee (NRERC).

Result: Overalll, an increased fold change in the mRNA expression of TLRs was observed in latently infected individuals relative to non-infected ones. Remarkable fold change was observed in TLR-2 and TLR-6 in TST positive individuals relative to TST negatives (mean with SEM) whereas a slight fold decrease was observed for TLR-1 gene. On the other hand, we found a strong positive linear correlation between intra compartment (TLR9) receptors and surface receptors TLRs (TLR1, TLR2, TLR4, and TLR6) expression in latent infected tuberculosis relative to non-infected. Similarly, the relative expression of TLR mRNAs was compared between children and adolescents and both sexes. However, no apparent difference was observed in the fold change expression of TLRs across age and sex.

Conclusions:Our results suggest that the up-regulation of TLR-2 TLR-4, TLR-6, and TLR-9 genes have role in latent tuberculosis infection through TLRs signalling a pathway and synergic effect with other immunological markers to maintain and continuous stimulation of immune response against latent tuberculosis in human. Therefore, further studies are needed, in order to elucidate whether those genes have used as booster vaccine or therapeutic agents and could serve as a specific molecular marker of latent TB.

Keywords: Tuberculosis, Innate immunity, Latency, Age, Toll-like receptors, Children, Adolescence