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      Phosphodiesterase-4 Inhibition Alters Gene Expression and Improves Isoniazid – Mediated Clearance of Mycobacterium tuberculosis in Rabbit Lungs

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          Abstract

          Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis ( Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.

          Author Summary

          Tuberculosis (TB) caused by Mycobacterium tuberculosis ( Mtb) is a leading infectious cause of morbidity and mortality. Although current antibiotic regimens can cure TB, treatment requires at least six months for completion. Recent studies indicate that bacteria in a less metabolically active state are less responsive to antibiotic killing and suggest that this may partly explain the long duration required for TB treatment. In this study, using a rabbit model of pulmonary TB, we show that immune modulation of Mtb infected animals with CC-3052, a phosphodiesterase-4 (PDE4) inhibitor that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP levels, resulted in the down-regulation of host genes involved in the innate immune response. Bacteria from the lungs of CC-3052 treated rabbits displayed differential expression of those genes associated with stress responses. In addition, co-treatment of INH with CC-3052 abolished the INH-induced Mtb gene expression in the infected rabbits. Importantly, bacillary clearance from the lungs of rabbits co-treated with CC-3052 and INH was improved over that in animals treated with INH alone. The results of this study provide a basis for novel use of immune modulation to improve the efficacy of antibiotic therapy and to shorten the duration of TB treatment.

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          Most cited references104

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          Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice.

          Understanding the immunological mechanisms of protection and pathogenesis in tuberculosis remains problematic. We have examined the extent to which tumor necrosis factor-alpha (TNF alpha) contributes to this disease using murine models in which the action of TNF alpha is inhibited. TNF alpha was neutralized in vivo by monoclonal antibody; in addition, a mouse strain with a disruption in the gene for the 55 kDa TNF receptor was used. The data from both models established that TNF alpha and the 55 kDa TNF receptor are essential for protection against tuberculosis in mice, and for reactive nitrogen production by macrophages early in infection. Granulomas were formed in equal numbers in control and experimental mice, but necrosis was observed only in mice deficient in TNF alpha or TNF receptor. TNF alpha and the 55 kDa TNF receptor are necessary conditions for protection against murine M. tuberculosis infection, but are not solely responsible for the tissue damage observed.
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            Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis.

            Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha-crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.
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              Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism.

              Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multidrug-tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug or more specific efflux pump inhibitors to standard antitubercular therapy should shorten the duration of curative treatment. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                September 2011
                September 2011
                15 September 2011
                : 7
                : 9
                : e1002262
                Affiliations
                [1 ]Laboratory of Mycobacterial Immunity and Pathogenesis, the Public Health Research Institute (PHRI) at the University of Medicine and Dentistry of New Jersey (UMDNJ), Newark, New Jersey, United States of America
                [2 ]Biological Sciences Department, New York City College of Technology, Brooklyn, New York, United States of America
                [3 ]Novartis Institute for Tropical Diseases, Singapore
                [4 ]Celgene Corporation, Summit, New Jersey, United States of America
                Harvard School of Public Health, United States of America
                Author notes

                Conceived and designed the experiments: SS DF LT GK. Performed the experiments: SS PO LT GY MK VD BP. Analyzed the data: SS GK. Contributed reagents/materials/analysis tools: VD GM. Wrote the paper: SS DF LT GK.

                Article
                PPATHOGENS-D-11-00516
                10.1371/journal.ppat.1002262
                3174258
                21949656
                9cfdd76c-1b43-48ee-b42b-13afc2b44bc3
                Subbian et al. 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
                : 9 March 2011
                : 22 July 2011
                Page count
                Pages: 19
                Categories
                Research Article
                Biology
                Genomics
                Functional Genomics
                Pharmacogenomics
                Immunology
                Genetics of the Immune System
                Immune Cells
                Immune Response
                Immune System
                Immunity
                Immunologic Subspecialties
                Immunomodulation
                Immunopathology
                Microbiology
                Bacteriology
                Bacterial Physiology
                Bacterial Pathogens
                Host-Pathogen Interaction
                Immunity
                Medical Microbiology
                Microbial Metabolism
                Microbial Pathogens
                Microbial Physiology
                Molecular Cell Biology
                Cellular Stress Responses
                Cellular Types
                Gene Expression
                Chemistry
                Medicinal Chemistry
                Medicine
                Clinical Immunology
                Drugs and Devices
                Infectious Diseases
                Pulmonology
                Veterinary Science
                Veterinary Pathology
                Veterinary Pharmacology

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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