ABSTRACT
INTRODUCTION: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. METHODS: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. RESULTS: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. CONCLUSION: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.
Subject(s)
Bacterial Toxins/pharmacology , Endotoxins/pharmacology , Escherichia coli Proteins/pharmacology , Free Radical Scavengers/antagonists & inhibitors , Immunosuppressive Agents/pharmacology , Interferon-gamma/antagonists & inhibitors , Macrophage Activation/drug effects , Nitric Oxide/antagonists & inhibitors , Animals , Cell Cycle/drug effects , Cell Line , Cell Shape/drug effects , Cell Survival/drug effects , Fibroblasts/drug effects , Interleukin-12/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred Strains , Toll-Like Receptor 2/drug effectsABSTRACT
Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E(2) (PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE(2) in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.
Subject(s)
Dendritic Cells/metabolism , Dinoprostone/biosynthesis , Fas Ligand Protein/genetics , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Myeloid Differentiation Factor 88/metabolism , Toll-Like Receptor 4/metabolism , Animals , CD3 Complex/metabolism , Cell Death/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoprotection/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , Fas Ligand Protein/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Macrophage Activation/drug effects , Macrophages/cytology , Macrophages/drug effects , Mice , Receptors, Prostaglandin E/metabolism , Receptors, Prostaglandin E, EP2 Subtype , Receptors, Prostaglandin E, EP4 Subtype , Signal Transduction/drug effects , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Up-Regulation/drug effectsABSTRACT
An 8-year-old son (L.A.S.) of consanguineous parents, presented recurrent bacterial infections, vasculitis and extremely low levels of serum C3 (0.15 microg/ml). The classical and alternative pathway haemolytic activities and the generation of opsonins and chemotactic factors derived from the activation of the complement system were markedly affected in the proband's serum. An in vitro addition of purified C3 restored the classical pathway-dependent haemolytic activity of his serum. Autoradiographs of the proband's lipopolysaccharide (LPS)-stimulated and 35S-labelled fibroblast supernatants after that the SDS-PAGE revealed no C3 alpha or beta chains. The amount of C3 mRNA synthesized by the proband's fibroblasts, as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) assays, was greatly reduced.
