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      Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of Mycobacterium tuberculosis in Human Macrophages

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          Abstract

          Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis ( MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

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          Most cited references57

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          High-efficiency transformation of mammalian cells by plasmid DNA.

          We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
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            Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors.

            The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.
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              Human IRGM induces autophagy to eliminate intracellular mycobacteria.

              Immunity-related p47 guanosine triphosphatases (IRG) play a role in defense against intracellular pathogens. We found that the murine Irgm1 (LRG-47) guanosine triphosphatase induced autophagy and generated large autolysosomal organelles as a mechanism for the elimination of intracellular Mycobacterium tuberculosis. We also identified a function for a human IRG protein in the control of intracellular pathogens and report that the human Irgm1 ortholog, IRGM, plays a role in autophagy and in the reduction of intracellular bacillary load.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                25 April 2013
                : 8
                : 4
                : e61925
                Affiliations
                [1 ]Department of Medicine, Denver Veterans Affairs Medical Center, Denver, Colorado, United States of America
                [2 ]Departments of Medicine and Academic Affairs, National Jewish Health, Denver, Colorado, United States of America
                [3 ]Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, United States of America
                [4 ]Denver Health Medical Center, Denver, Colorado, United States of America
                [5 ]Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, United States of America
                [6 ]Department of Medicine, Tri-Service General Hospital; National Defense Medical Center, Taipei, Taiwan
                [7 ]Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America
                Fundação Oswaldo Cruz, Brazil
                Author notes

                Competing Interests: The Authors have declared that no competing interests exist.

                Conceived and designed the experiments: XB EDC. Performed the experiments: XB NEF KC MTM SM. Analyzed the data: XB ARO WLS MJS MN EDC. Contributed reagents/materials/analysis tools: L. Gaido DP L. Griffin JRH REO. Wrote the paper: XB ARO L. Griffin JRH WHK DRV DJO EDC.

                Article
                PONE-D-12-30305
                10.1371/journal.pone.0061925
                3636238
                23634218
                08337319-737b-4744-9896-02f5e42fbbaa
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 2 October 2012
                : 14 March 2013
                Page count
                Pages: 13
                Funding
                This work was supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development 1 I01 BX001028-01A2 (to EDC), National Institutes of Health (NIH) PHL073907 (to DRV), and General Clinical Research Center Program Grant No. M01-RR00051. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Proteins
                Immune System Proteins
                Immunochemistry
                Immunology
                Immunity
                Immune Defense
                Immune Response
                Microbiology
                Immunity
                Immune Defense
                Molecular Cell Biology
                Signal Transduction
                Signaling in Selected Disciplines
                Immunological Signaling
                Medicine
                Clinical Immunology
                Immunity
                Immune Defense
                Infectious Diseases
                Bacterial Diseases
                Mycobacterium
                Tuberculosis
                Tropical Diseases (Non-Neglected)
                Tuberculosis
                Infectious Disease Control

                Uncategorized
                Uncategorized

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