Comparative gene expression in different stages of Mycobacterium tuberculosis infection in mice

Abstract

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. One-third of the world's population is estimated to be latently infected with M. tuberculosis; however, only approximately 10% of latently infected people will develop this overt disease, and their aerosol droplets can be transmitted through the air. Current tests available for the diagnosis of TB are limited by their inability to differentiate between latent tuberculosis infection (LTBI) and active TB disease. Therefore, an accurate and effective marker for differential diagnosis should be developed for predicting disease progression. Given the increasing interest in identifying new biomarkers against TB, we examined promising biomarkers that could distinguish between chronic/reactivation TB disease and LTBI using an M. tuberculosis-infected mouse model. The lung tissue and serum of C57BL/6 mice were used to identify the differences of gene expression among healthy, LTBI, chronic TB, spontaneous reactivation, and immunosuppressive drug-treated groups. First, microarray analysis using isolated mRNAs from mice tissues showed several important immune-related genes with different expression levels between the chronic/latent and healthy control groups (p < 0.05). Next, quantitative reverse transcription-polymerase chain reaction to validate the results obtained by the microarray was performed. Based on the cDNA microarray results, 17 candidate genes were selected and clustered into four groups: 1) chemokines excluding monocyte chemoattractant proteins (MCPs) (CXCL9, CXCL10, CXCL11, CCL5, CCL19); 2) MCPs (CCL2, CCL7, CCL8, CCL12); 3) Receptors (IL2Rβ, IL7R, IL12Rβ1, IL12Rβ2, IL21R, IL27Rα); and 4) TNF and IFN- genes. Results from the cDNA microarray and quantitative RT-PCR analyses revealed that expression of the selected cytokine genes was significantly higher in lung tissues of the chronic stage than of the latent stage. CXCL9, CCL7, CCL12 were noticeably increased in the chronic stage compared with those in the latent stage. Therefore, these three significantly increased cytokines in lung tissue from the mouse TB model might be candidates for biomarkers that distinguish the two disease stages. This information can be combined with already reported potential biomarkers to construct a network of more efficient TB markers. We used Luminex assays to confirm the same tendency of gene expression in mice serum. Second, there is a substantial need for biomarkers to distinguish latent, chronic TB from spontaneous reactivation, for predicting disease progression. To identify the immunological status of the latent, chronic, and reactivation stages, immunological genes were analysed in lung tissues from mice infected with M. tuberculosis. Gene expression was screened using cDNA microarray analysis and confirmed by quantitative RT-PCR using isolated microRNAs from mice tissues. In the result, 10 microRNAs (mmu-miR-1a-3p, mmu-miR-133a-5p, mmu-miR-133a-3p, mmu-miR-206-3p, mmu-miR-133b-3p, mmu-miR-3064-5p, mmu-miR-450b-3p, mmu-miR-26b-3p, mmu-miR-181a-2-3p, mmu-miR-8114) were likely to be the biomarkers that can distinguish between latent and reactivation TB. Specifically, mmu-miR-206-3p was noticeably increased in the reactivation stage compared with the latent and chronic stage. And mmu-miR-1a-3p is expected to be a biomarker candidate that can simultaneously distinguish latent, chronic, and reactivation stages. In conclusion, these findings suggest that the protective mechanisms against TB infection may be related to chemokines, MCPs, chemokine receptors and microRNAs that modulate the activity of immune responses, and some of which are potential biomarkers distinguishing different stages of M. tuberculosis infection.

결핵의 원인은 대부분 Mycobacterium tuberculosis 이며, 감염성 질병 중에서 사망률이 세계 2위에 랭크되어 있는 심각한 질병이다. WHO는 2013년까지 M. tuberculosis 에 감염되어 발병된 사람은 900만 명, 사망자는 150만 명이라고 밝혔다. 현재 전체 인구의 약 1/3, 즉 20억 명 이상이 TB 잠복 감염자인 것이다. 그 중 5-10% 만이 활동성 결핵 환자가 되는데, 대부분 ...