Small RNA Profiles of Serum Exosomes Derived from Individuals with Latent and Active Tuberculosis
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ABSTRACT: We performed small RNA sequencing to explore small RNA profiles of serum exosomes derived from LTBI and TB patients and healthy controls (HC). Our results revealed distinct miRNA profile of the exosomes from the three samples. We identified many differentially expressed miRNAs, including some specifically expressed miRNAs in the three samples. Besides the specially expressed miRNAs, we demonstrated distinct expression panels of the serum exosomal miRNAs from LTBI and TB samples, and six expression patterns among the three samples. These specifically expressed miRNAs and differentially expressed miRNAs in different panels and patterns provide potential biomarkers for detection/diagnosis of latent and active TB using exosomal miRNAs. Additionally, we also discovered plenty of small RNAs derived from genomic repetitive sequences (e.g., SINEs, LINEs and LTR), which might play roles in host immune responses along with Mtb infection progresses. Overall, our findings provide important reference and improved understanding about miRNAs and repetitive region-derived small RNAs in exosome during Mtb infectious process, and facilitate the development of potential molecular targets for detection/diagnosis of latent and active tuberculosis.
Project description:Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. This study intends to explore the potential function of miRNAs in the interaction of macrophages with Mtb Hsp16.3 and provide insights for investigating the role of macrophage homeostasis in LTBI. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI.
Project description:Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. This study intends to explore the potential function of miRNAs in the interaction of macrophages with Mtb Hsp16.3 and provide insights for investigating the role of macrophage homeostasis in LTBI.
Project description:the microRNA profiles of the host macrophages were studied by microarray in a small cohort with active MTB disease, latent infection (LTBI), and from healthy controls. From each individual in the three cohorts: the healthy (n=3), the latent (n=4), and the active TB patients (n=3), whole blood specimens were collected for monocytes isolation. Monocytes were induced into macrophage in vitro and total RNA were extracted for miRNA profiles analysis.
Project description:Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune competent individuals in absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by positive immunologic response to Mtb antigens with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered mainly by an inability to achieve complete bacillary clearance. We have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathology occurs spontaneously. The evidence that Mtb-CDC1551-infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated following immune suppression. The microarray experiments involves comparison of: 1) Changes in rabbit gene expression between Mtb-CDC1551 infected and uninfected animals at 2,4,8 and 12 weeks post infection. New Zealand White rabbits were infected with Mtb CDC1551 at 3.5log10 (on day 0). Lung tissue from Mtb-infected rabbits were isolated from uninfected (control) and at 2, 4, 8 and 16 weeks post infection and used for total RNA extraction. Total rabbit lung RNA was used for microarray analysis to determine infection induced changes in host gene expression.
Project description:Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world’s population. The majority of infected persons are asymptomatic, but latent TB infection (LTBI) can progress to active clinical disease in 5-10% of infected individuals. The immune mechanisms that govern progression from latent to active pulmonary TB (PTB) remain poorly defined. An in-depth understanding of immune factors correlating with TB disease, as well as protection during TB, is necessary for developing new immunotherapies to promote immune control of Mtb. Experimentally Mtb-infected non-human primates (NHP) mirror the disease progression and pathology observed in humans and can recapitulate both PTB and LTBI. In the present study, we have characterized the lung immune landscape in NHPs with LTBI and PTB using high-throughput technologies including single-cell RNA sequencing (scRNA-seq) and Time of flight cytometry (CyTOF). We show that the three defining features of PTB in macaque lungs are the influx of plasmacytoid DCs (pDCs), an Interferon (IFN)-exhibiting alveolar macrophage population and predominant activated T cell responses. These features contribute to uncontrolled inflammation and disease without mediating Mtb control. In contrast, a CD27+ Natural killer (NK) cell subset accumulated in the lungs of LTBI macaques and in circulation in individuals with LTBI, thus providing novel insights into the protective lung landscape that functions during TB latency. A comprehensive understanding of the lung immune landscape as described here will improve our overall understanding of TB disease immunopathogenesis and provide novel targets for design of new therapies and vaccines for TB control.
Project description:Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune competent individuals in absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by positive immunologic response to Mtb antigens with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered mainly by an inability to achieve complete bacillary clearance. We have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathology occurs spontaneously. The evidence that Mtb-CDC1551-infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated following immune suppression. The microarray experiments involves comparison of: 1) Changes in rabbit gene expression between Mtb-CDC1551 infected and uninfected animals at 2,4,8 and 12 weeks post infection.
Project description:To further understand the host immune factors involved in the progression from latent tuberculosis infection (LTBI) to active tuberculosis (TB) and identify the potential signatures for discriminating TB from LTBI, the genome-wide transcriptional profile of the Mycobacterium.Tuberculosis (M.TB)–specific antigens stimulated peripheral blood mononuclear cells (PBMCs) from active TB, LTBI and health controls (HCs) were performed. A total of 209- and 234- differentially expressed genes (Fold change > 4, P < 0.05) were detected in comparisons of TB vs. LTBI and TB vs. HCs, respectively. Nineteen differentially expressed genes with top fold change between TB and the other two groups were validated in the same RNA samples by real-time PCR, and showed 94.7% consistent expression pattern with microarray test.
Project description:We performed RNA-seq analysis on exosomes derived from the clinical specimens of healthy control (HC), active tuberculosis (ATB) and latent tuberculosis infection (LTBI) individuals. Our results revealed the distinguished gene expression panels and patterns of the exosomes for the LTBI and ATB patients: We identified many up-regulated and down-regulated differentially expressed genes (DEGs) in the LTBI and ATB samples, and further screened the top-20 DEGs, which might provide a clue to differentiate HC, LTBI and ATB; We classified all the DEGs into six expression patterns, screened the top-20 genes in each pattern, and mainly focused on those highly expressed in LTBI and ATB; A lot of Mtb genes were only expressed and enriched in the exosomes of LTBI patients; Pathway and functional analysis further indicated the gradually increased deteriorated healthy signals in LTBI and ATB samples, including down-regulated signaling pathways/immune response, and up-regulated apoptosis/necrosis. Our findings not only add new data to tuberculosis clinical studies, but also facilitate the development of potential targets for the diagnosis, prevention and treatment of tuberculosis.
Project description:Increasing evidence supports a role ofthat antibodies in thecan defense protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially at in the mucosal airways level, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses that were associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) compared to uncontrolled infection (TB) in nonhuman primates. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to and two- and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays
Project description:Increasing evidence supports a role ofthat antibodies in thecan defense protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially at in the mucosal airways level, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses that were associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) compared to uncontrolled infection (TB) in nonhuman primates. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to and two- and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays