Transcription profiling of responses to TNF-alpha, IL-1beta, peptidoglycan or lipopolysaccharide (TLR2/4 agonists) evaluated at 4 hrs in peripheral blood monocytes (PBM) from the patient bearing the IRAK4 Q293X mutation vs PBM from 5 healthy individuals
ABSTRACT: IL-1R associated kinase-4 (IRAK4) is a key enzyme required for activation of the common Toll-like Receptor (TLR) signaling cascade, which results in the transcription of inflammatory and immunity genes. IRAK-4 deficiency has recently been described as a rare form of innate immunodeficiency. Patients present with pyogenic bacterial infections and bacteraemia, particularly with Gram+ Streptococcus pneumoniae, and isolated PBMC from these patients fail to produce inflammatory cytokines in response to TLR agonists. We embarked on this study for several purposes: (1) to identify defective gene response resulting from IRAK4-deficiency that are responsible for the patients' susceptibility to infection by particular bacteria (2) to identify genetic responses that confer relatively normal immunity in these patients despite having a defect in such a critical component of the innate immune system (3) to gain understanding of the transcriptional regulation of inflammatory genes (4) to gain insight into TLR signal transduction pathways, in particular, the role of IRAK4 in the TLR2 and TLR4 pathways initiated by Gram+ and Gram- bacterial components respectively. Transcriptional responses to TNF-alpha, IL-1beta, peptidoglycan or lipopolysaccharide (TLR2/4 agonists) were evaluated at 4 hrs in peripheral blood monocytes (PBM) from the patient bearing the IRAK4 Q293X mutation compared to PBM from 5 healthy individuals.
BACKGROUND:Activation of Toll-like receptors (TLRs) is widely accepted as an essential event for defence against infection. Many TLRs utilize a common signalling pathway that relies on activation of the kinase IRAK4 and the transcription factor NFkappaB for the rapid expression of immunity genes. METHODS:21 K DNA microarray technology was used to evaluate LPS-induced (TLR4) gene responses in blood monocytes from a child with an IRAK4-deficiency. In vitro responsiveness to LPS was confirmed by re ...[more]
Project description:Human bronchial epithelial cells (16HBE14O-) were stimulated with the commensal bacterium S. salivarius K12, or the pathogens S. aureus, P. aeruginosa, or S. enteritidis (subtype Typhimurium) for 1 hour.
Project description:The objective of the study was to discover new functions of chemokine CXCL9/MIG, a cationic chemokine that has antimicrobial properties. Human monocytic cell line, THP-1 cells were stimulated 4 hours with chemokine MIG or CCL2/MCP-1 (a chemokine that is not positively charged and has no antimicrobial activity). By using a 21,000 oligo-based DNA human microarray we analyzed gene expression profiles induced by MIG and by MCP-1. The gene expression profile induced by MIG was >85% different from that induced by MCP-1. MIG increased transcription of genes encoding various chemokines (including CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL2/MCP-1 and CXCL8/IL-8), cytokines (TNF-alpha), indicating that MIG has an immunomodulatory effect on THP-1 cells. Additionally, MIG strongly up-regulated a set of genes identified as having tumor suppressor functions, including BTG2, BTG1, P21, IGFBP3, DSCR1 and genes encoding markers for mature leukocyte differentiation (PLAU, LPL, PLAUR etc). In parallel, MIG down-regulated the expression of oncogenes (MYC), and genes related to cell proliferation and cell cycle progression. This gene profile strongly suggested that the chemokine MIG had anti-proliferative activity and might induce THP-1 cells to undergo terminal differentiation.
Project description:The objective of the study was to evaluate transcriptional response of endotoxin-stimulated human monocytic cells in presence or absence of host defense peptide LL-37 at low physiologically relevant concentrations. Human monocytic cells THP-1 were stimulated with LPS (10ng/ml) in presence or absence of LL-37 (5 ug/ml), as well as with the peptide alone, for 4 hours. The trends of LPS-induced altered gene expression in presence of the peptide were further validated by quantitative real-time PCR.
Project description:The objective of the study was to evaluate transcriptional response of endotoxin-stimulated human monocytic cells in presence and absence of host defense peptide LL-37. A functional genomics approach was used to establish a temporal transcriptional profile and identify differentially expressed genes in LPS (100ng/ml)-stimulated human monocytic THP-1 cells, in the presence or absence of LL-37 (20ug/ml) after 1, 2, 4 and 24 hrs of stimulation. The peptide significantly inhibited the expression of LPS-induced pro-inflammatory genes regulated by NF-kappa B, such as NF-kappa B1 (p105/p50) and TNF-alpha-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-alpha-induced protein 3 (TNFAIP3) and the NF-kappa B inhibitor, NF-kappa BIA, or genes involved in cell movement and recruitment (chemokines). The trends of gene expression were further validated by quantitative real-time PCR. This study implicates that LL-37 plays a role in the delicate balancing act of inflammatory responses.
Project description:Persons with Chronic Granulomatous Disease, are susceptible to a narrow range of infections resulting from the failure of cells to generate toxic reactive oxygen species (ROS) required for phagocytice oxidative killing of microorganisms. For unknown reasons, many inflammatory conditions (inflammatory bowel disease, periodontal inflammation, granulomatous obstruction of the urinary and gastrointestinal tract and “sterile” inflammation of the lungs and other organs) are also associated with the disease. The goal of this study is to investigate the role of ROS in the regulation of inflammatory and immunity genes induced by Toll-like Receptors (TLRs). Transcriptional responses to peptidoglycan or lipopolysaccharide (TLR2/4 agonists) were evaluated at 4 and 24 hrs in peripheral blood monocytes (PBM) from three males with CGD compared to PBM from 5 healthy individuals.
Project description:Effect of peptide IDR-1 on CD14+ human monocytes. >br< >br< Effect of peptide IDR-1 on LPS-induced responses in CD14+ human monocytes.>br< >br< Human peripheral blood mononuclear cells (PBMC) were treated with innate defence regulator peptide IDR-1 in the presence and absence of LPS (purified from Pseudomonas aeruginosa) for 4hrs. The objective was to obtain a transcriptional profile of responses induced by IDR-1 and the effect of IDR-1 on LPS-induced transcription.
Project description:To assess the credibility of our experimental data in E-TABM-327 we compared transcriptional profiiles of media shifted Aspergillus fumigatus cultures using RNA that was subjected to 1, 2 or no rounds of amplification. The protocol dependent changes in log ratios were assessed
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Project description:The aim of this study was to identify alarm (fast) and acclimation phase (delayed) changes in the gene expression pattern in leaves of maize (CM 109 genotype) subjected to moderate chilling for 28 hours.