Project description:Background: Dissecting the in-vivo host-pathogen interplay is crucial in understanding the molecular mechanisms governing control or progression of the infection. While Dual RNA-seq offers significant advantages over traditional approaches in the analysis of intracellular infections, to date technical challenges have restricted the application of this technology predominantly to tissue culture infection models. We addressed this problem by developing new protocols which allowed us to perform, for the first time, Dual RNA-seq on Mycobacterium tuberculosis-infected and ontogenetically distinct macrophages isolated directly from murine lungs. Results: Through analysis of the Mtb transcripts specifically induced during infection of alveolar and interstitial macrophages, we first identified an in-vivo signature, a set of 180 genes uniquely upregulated by Mtb in mouse lung macrophages. Furthermore, comparative analysis of transcripts from both Mtb and the two macrophage lineages uncovers that alveolar macrophages exhibit an M2-like polarization profile and promote bacterial growth through increased access to iron and fatty acids, while the interstitial macrophages exhibit a pro-inflammatory profile and restrict bacterial growth through iron sequestration and higher levels of nitric oxide. Conclusions: Our study reports a new bacterial transcripts enrichment protocol which enabled us to perform dual-RNA seq at the infected cell level, providing a comprehensive look at the divergent transcriptional signatures that characterizes survival of Mtb in the two major myeloid cells. The data re-emphasizes the significance of nutritional immunity for in vivo control of infection via an unbiased interrogation of both host and pathogen transcriptomes. Our approach provides a new tool to probe bacterial physiology at the host cell level in an in-vivo environment and therefore has a broad impact in understanding infections of intracellular pathogens.
Project description:The purpose of this study was to identify Mtb- and hsa-encoded miRNAs produced in infected macrophages. RNA from 9 THP-1 samples (3 were uninfected, 3 were infected with Mtb H37Rv for 3 days and 3 were infected with Mtb H37Rv for 6 days) was sequenced and miRNAs were detected.
Project description:The success of Mycobacterium tuberculosis (Mtb) as pathogen is tightly linked to its ability to recalibrate host metabolic processes in infected host macrophages. Correspondingly, analysis of RNA-sequencing datasets showed altered gene expression of key metabolic enzymes involved in NAD+, creatine, glucose and glutamine metabolism (e.g NAMPT, IDO1, SLC6A8, CKB, HK2) in Mtb-infected M2 macrophages.
Project description:Primary human monocytes were isolated from four healthy human blood donors. Monocytes were isolated from PBMC buffy coats using plastic adherence for 4 hours. Monocytes were allowed to differentiate into macrophages over a period of 1 week. Macrophages from each of the 4 donors were split into two groups - uninfected and infected with Mycobacterium tuberculosis (Mtb). Cells in the infection group were infected with Mtb for 48 hours at a multiplicity of infection (MOI) of 10. Following incubation, uninfected and infected cells were harvested for RNA. RNA was used for next-generation RNA sequencing. Raw RNA sequencing data was processed using a HISAT2, Stringtie, Ballgown, DESeq2 pipeline. Processed data was used to measure differential expression between uninfected and infected macrophages.
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:The peritoneal macrophages were infected with Mtb H37Rv for 4 hours, and the miRNA expression profile were analyzed with deep sequencing.
Project description:Primary human monocytes were isolated from four healthy donors. Monocytes were differentiated into macrophages, infected with virulent Mycobacterium tuberculosis (Mtb), strain H37Rv, for 24 or 48 hours, at a multiplicity of infection of 5 or 10. Following infection, infected cells and time-matched uninfected controls were harvested, total RNA including small RNAs was isolated and used for next-generation small RNA sequencing. Small RNA sequencing data was processed using miRge2.0, including a novel miRge2.0-based tRF detection tool. Processed data was used to determine differential expression of microRNAs and differential production of tRNA-derived fragments (tRFs) during infection with Mtb.
2020-10-15 | GSE151050 | GEO
Project description:RNA-seq for MTB infected mice BMDMs
Project description:Transcriptional profiling of RNA samples from un-infected Rhesus Bone Marrow Derived Macrophages (RhBMDMs) exposed to IMDM complete media used as control were compared to RNA from RhBMDMs infected for 24 hr with 'dos' mutants of Mtb viz. MtbΔdosR, MtbΔdosS, or MtbΔdosT
