Project description:The goal of this experiment was to determine if macrophages can distinguish between M. tuberculosis grown in standard, zinc-replete media and those grown in zinc-limited media. In order to determine this, we performed RNA-sequencing (RNA-seq) of RAW 264.7 macrophages infected with zinc-limited Mtb and zinc-replete Mtb at 4 hours post-infection (hpi) and 24hpi. Transcriptional response in the infected macrophages was compared to uninfected controls (macrophages exposed to growth media alone) and between macrophages infected with zinc-limited Mtb and zinc-replete Mtb at each time point.
Project description:CD4+ T cell-mediated control of tuberculosis (TB) requires recognition of macrophages infected with Mycobacterium tuberculosis (Mtb). Yet not all Mtb-specific T cells recognize infected macrophages. Using infected monocyte-derived macrophages (MDMs) and autologous memory CD4+ T cells from individuals with latent Mtb infection (LTBI), we isolate and quantify CD4+ T cells activated in response to infected macrophages. We use T cell antigen receptor (TCR) sequencing to determine the total and unique Mtb-specific TCR clonotypes linked to recognition of infected macrophages, and find that a subset required exogenous antigen exposure, suggesting incomplete recognition. Clonotypes specific for multiple Mtb antigens, and other pathogens, were also identified. We use bulk TCRb deep sequencing from total CD4+ T cells isolated from 10x106 PBMCs from each participant to determine the natural circulating frequencies of relevant TCR clonotypes. We used single-cell RNA sequencing (scRNAseq) to examine the effector functions of CD4+ T cells in response to infected macrophages. Mtb-specific clonotypes expressed signature effector functions dominated by IFNg, TNF, IL-2, and GM-CSF or chemokine production and signaling. We propose TB vaccines that elicit T cells specific for T7SS substrates, recognize infected macrophages, and express canonical effector functions will offer protection against TB.
Project description:<p>Regulatory T cells (Tregs) expand during Mycobacterium tuberculosis (Mtb) infection and suppress T cell mediated control. Whether Mtb actively contributes to this process is unclear. Here, using a genome-wide mutant library, we show that expression of Mtb Rv1272c, an ATP-binding cassette transporter, increased under hypoxic condition, promotes Mtb survival in vivo by increasing lecithin import, followed by the production and release of linoleic acid. Linoleic acid released by infected macrophages promoted surface trafficking of the immune checkpoint molecule CTLA-4 in Tregs via the Ca²⁺ transporter ATP2a3. This in turn inhibited macrophage reactive oxygen species production and promoted Mtb survival inside macrophages. Rv1272c-induecd linoleic acid further promoted Mtb immune evasion via increasing CTLA-4 surface trafficking on Tregs in vivo. [AU please mention in vivo work on virulence as well] Mechanistically, linoleic acid interacts with ATP2a3 in Tregs and promotes mitochondria-associated endoplasmic reticulum (ER) membrane formation. This facilitates ER to mitochondrial Ca2+ transfer, depletion of Ca2+ in the ER, and triggers store-operated calcium entry, thus elevating cytosolic Ca2+ levels to increase Ca2+-dependent CTLA-4 surface trafficking in Tregs. These findings reveal that Mtb can use a metabolite to manipulate host responses and promote its intracellular survival.</p>
Project description:Host macrophage transcriptional responses to intracellular pathogens remain poorly characterized. We screened transcriptional enhancers engaged in response to M. tuberculosis (Mtb) infection by ChIPseq analysis of histone H3 lysine 4 monomethylation (H3K4me1). De novo monomethylation during infection was associated with genes implicated in host defense and apoptosis. These regions were enriched for binding sites for ETS transcription factor family members and response elements for nuclear receptors, including liver X receptors (LXRs) and peroxisomal proliferator activated receptors (PPARs), many of which were encompassed by transposable elements. LXRa expression was strongly induced by infection, whereas that of PPARs was unaffected. LXR DNA binding and NCoR corepressor recruitment increased proportionately in infected cells but coactivator association was unchanged, consistent with a lack of induction of endogenous agonists. However, treatment of infected cells with LXR agonist T0901317 strongly increased coactivator recruitment and induced a gene expression program characterized by enhanced innate immune signaling and lipid metabolism. Remarkably, T0901317 treatment selectively induced apoptosis in infected macrophages, and was accompanied by Mtb death, reducing mycobacterial burden 18-fold relative to vehicle 5d after infection. These studies define macrophage transcriptional responses to Mtb infection, and suggest that tissue-specific LXRa agonists may be efficacious in clinical management of tuberculosis.
Project description:The aim of this study was to measure the immune response of alveolar macrophages (AMs) to intracellular Mtb infection in vivo. We characterized the transcriptional profile of murine Mtb-infected AMs after aerosol infection by sorting cells from bronchoalveolar lavage fluid and performing low-input RNA-sequencing. To determine the effect of the transcription factor Nrf2 on this response we also measured the transcriptional profiles of AMs from mice lacking Nrf2.
Project description:How the complex interaction between Mycobacterium tuberculosis (Mtb) and the host is regulated during infection is still not well understood. Using a systems biology approach, we demonstrate here that miR-155 is one of several microRNAs that regulate host gene expression over the first 48 hours of Mtb infection in macrophages. miR-155 regulates the cell survival of Mtb-infected macrophages through SHIP1/AKT signaling. Using timecourse gene expression data, we constructed a miRNA regulatory network for the innate immune response to Mtb infection by WT macrophages. The network suggested a role for seven miRNAs in regulating the host response to Mtb, with miR-155 being one of them. We then validated a role for miR-155 by comparing the response between WT and miR-155-/- macrophages.
Project description:How the complex interaction between Mycobacterium tuberculosis (Mtb) and the host is regulated during infection is still not well understood. Using a systems biology approach, we demonstrate here that miR-155 is one of several microRNAs that regulate host gene expression over the first 48 hours of Mtb infection in macrophages. miR-155 regulates the cell survival of Mtb-infected macrophages through SHIP1/AKT signaling. Using timecourse gene expression data, we constructed a miRNA regulatory network for the innate immune response to Mtb infection by WT macrophages. The network suggested a role for seven miRNAs in regulating the host response to Mtb, with miR-155 being one of them. We then validated a role for miR-155 by comparing the response between WT and miR-155-/- 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:Host macrophage transcriptional responses to intracellular pathogens remain poorly characterized. We screened transcriptional enhancers engaged in response to M. tuberculosis (Mtb) infection by ChIPseq analysis of histone H3 lysine 4 monomethylation (H3K4me1). De novo monomethylation during infection was associated with genes implicated in host defense and apoptosis. These regions were enriched for binding sites for ETS transcription factor family members and response elements for nuclear receptors, including liver X receptors (LXRs) and peroxisomal proliferator activated receptors (PPARs), many of which were encompassed by transposable elements. LXRa expression was strongly induced by infection, whereas that of PPARs was unaffected. LXR DNA binding and NCoR corepressor recruitment increased proportionately in infected cells but coactivator association was unchanged, consistent with a lack of induction of endogenous agonists. However, treatment of infected cells with LXR agonist T0901317 strongly increased coactivator recruitment and induced a gene expression program characterized by enhanced innate immune signaling and lipid metabolism. Remarkably, T0901317 treatment selectively induced apoptosis in infected macrophages, and was accompanied by Mtb death, reducing mycobacterial burden 18-fold relative to vehicle 5d after infection. These studies define macrophage transcriptional responses to Mtb infection, and suggest that tissue-specific LXRa agonists may be efficacious in clinical management of tuberculosis.
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.