Project description:This experiment explored in vitro responses of monocytes following in vitro stimulation with purified protein derivative (PPD) for 4 hours. Monocytes were collected both from individuals with active tuberculosis (TB) disease, as well as those with latent TB infection.
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:Whole transcriptome profiling by microarray of monocytes stimulated with LPS and treated with trabectedin, lurbinectedin or doxorubicin
Project description:Tuberculosis remains a major cause of death from an infectious disease worldwide, yet only 10% of people infected with Mycobacterium tuberculosis develop disease. Defining both necessary and sufficient immunologic determinants of protection remains a great scientific challenge. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify human potential candidate markers of host defense by studying gene expression profiles of macrophages, cells which, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene co-expression network analysis revealed an association between the cytokine, IL-32, and the vitamin D antimicrobial pathway in a network of IFN-γ and IL-15 induced ‘defense response’ genes. IL-32 was sufficient for induction of the vitamin D-dependent antimicrobial peptides, cathelicidin and DEFB4, and generation of antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. The IL-15 induced ‘defense response’ macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent vs. active tuberculosis or healthy controls, and a co-expression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15 induced gene network. Inferring that maintaining M. tuberculosis in a latent state and preventing transition to active disease represents host resistance, we believe these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis. Adherent peripheral blood monuclear cells were derived by Ficoll-Hypaque from the whole blood of four healthy donors. Cells adhered to tissue culture-treated plates for 2 h in 1% Fetal Bovine Serum (FBS) in RPMI. Cells were washed then stimulated with IL-10 (10ng/ml), IL-15 (10ng/ml) (R&D Systems), or IL-4 (1U/ml) in RPMI 1640 supplemented with 10% FBS at 37°C, 5% CO2. Cells were harvested at 6 h and 24 h after stimulation and monocytes purified by CD14 microbeads (Miltenyi Biotec) for a confirmed monocyte purity of at least 90%. RNA from purified monocytes extracted by Trizol and purified by Qiagen RNeasy Kit. RNA probe and microarray performed by UCLA Clinical Microarray Core using Ambion labeling kit and Affymetrix Human U133 Plus 2.0 array.
Project description:As innate immune cells, monocytes play a central role in antifungal immunity. Using proteome studies in primary human monocytes, which were stimulated by Candida albicans (yeast) in vitro. Here we describe the changes of proteins in monocytes and demonstrate that in the early stage of infection, the differences of innate immune response triggered by C. albicans over time.
Project description:Tuberculosis (TB) is responsible for the majority of mortality and morbidity associated with infectious diseases worldwide. The characterization of exact molecular components of immune response associated with protection against TB may help design more effective therapeutic interventions. In this study, we aimed to characterize the immune signature of monocyte subsets associated with active versus latent infection with Mycobacterium tuberculosis. Transcriptomic profiling using RNA sequencing was performed on classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) monocytes isolated from individuals with active TB (at diagnosis and 2 months post treatment), latent TB, as well as from TB negative healthy controls. Overall, we found specific gene signatures for each monocyte subset that could successfully discriminate between individuals with active TB at diagnosis, treated active TB, latent TB and healthy controls.
Project description:Latent HIV-1 infection poses a major challenge in complete viral remission and cure. HIV-1 latency is a multi-dimensional, dynamic process and many aspects of how the viral latency is established and maintained still remains incompletely characterized. Here, we have investigated the host chromatin organization and transcriptomic changes in active- and latently-infected SupT1 cells. We employed an in vitro model of HIV-1 latency in SupT1 cells using a dual-reporter virus, HIVGKO, which enables high purity sorting and characterization of active- and latently-infected cells. We found a significant divergence in chromatin organization and gene expression pattern between active and latent infection compared to uninfected cells. Latent infection results in a repressive reorganization of the host chromatin, while active infection leads to an overall increase in chromatin accessibility. A stronger correlation was also observed between chromatin accessibility and gene expression in latent infection, which was manifested in a greater alteration of the cellular transcriptome in latent than active infection, for both proteincoding and long-non-coding RNAs (lncRNAs). We identified a number of novel lncRNAs associated with either active and latent infection. A reversal in expression pattern of latency-associated lncRNAs following PMA-induced reactivation indicated their infection state-specific expression and potential roles in HIV-1 latency. Taken together, this integrated, comparative study revealed that latent HIV-1 infection requires a substantially greater alteration in cellular epigenome and transcriptome. Understanding of the distinct cellular states conducive to active and latent infection may support devising strategies for specific modulation of host cellular functions as a curative intervention for HIV-1.
Project description:BACKGROUND: Combination antiretroviral therapy (cART) is able to control HIV-1 viral replication, however long-lived latent infection in resting memory CD4+ T-cells persist. The mechanisms for establishment and maintenance of latent infection in resting memory CD4+ T-cells remain unclear. Previously we have shown that HIV-1 infection of resting CD4+ T-cells co-cultured with CD11c+ myeloid dendritic cells (mDC) produced a population of non-proliferating T-cells with latent infection. Here we asked whether different antigen presenting cells (APC), including subpopulations of DC and monocytes, were able to induce post-integration latent infection in resting CD4+ T-cells, and examined potential cell interactions that may be involved using RNA-seq. RESULTS: mDC (CD1c+), SLAN+ DC and CD14+ monocytes were most efficient in stimulating proliferation of CD4+ T-cells during syngeneic culture and in generating post-integration latent infection in non-proliferating CD4+ T-cells following HIV-1 infection of APC-T-cell co-cultures. In comparison, plasmacytoid DC (pDC) and B-cells did not induce latent infection in APC-T-cell co-cultures. We compared the RNA expression profiles of APC subpopulations that could and could not induce latency in non-proliferating CD4+ T-cells. Gene expression analysis, comparing the mDC, SLAN+ DC and CD14+ monocyte subpopulations to pDC identified 53 upregulated genes that encode proteins expressed on the plasma membrane that could signal to CD4+ T-cells via cell-cell interactions (32 genes), immune checkpoints (IC) (5 genes), T-cell activation (9 genes), regulation of apoptosis (5 genes), antigen presentation (1 gene) and through unknown ligands (1 gene). CONCLUSIONS: APC subpopulations from the myeloid lineage, specifically mDC subpopulations and CD14+ monocytes, were able to efficiently induce post-integration HIV-1 latency in non-proliferating CD4+ T-cells in vitro. Inhibition of key pathways involved in mDC-T-cell interactions and HIV-1 latency may provide novel targets to eliminate HIV latency. mRNA profiles of sorted, pure antigen presenting cells including, CD1c+ myleoid dendirtic cells (mDC), SLAN+ mDC, CD14+ monocytes and plasmacytoid DC (pDC), were generated using next generation sequencing in triplicate, using Illumina Illumina Hiseq 2000.