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NF-Κb Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages

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NF-Κb Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages NF-κB Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages This information is current as Maximiliano Gabriel Gutierrez, Bibhuti B. Mishra, Luisa of September 27, 2021. Jordao, Edith Elliott, Elsa Anes and Gareth Griffiths J Immunol 2008; 181:2651-2663; ; doi: 10.4049/jimmunol.181.4.2651 http://www.jimmunol.org/content/181/4/2651 Downloaded from References This article cites 49 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/181/4/2651.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology NF-␬B Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages1 Maximiliano Gabriel Gutierrez,2,3* Bibhuti B. Mishra,2† Luisa Jordao,† Edith Elliott,‡ Elsa Anes,† and Gareth Griffiths* Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-␬B in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-␬B that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-␬B activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-␬B during infection. Our results argue that NF-␬B activation increases the synthesis of membrane trafficking molecules, which may Downloaded from be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-␬B inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-␬B in the innate immune response can now be expanded to include regulation of membrane trafficking during infection. The Journal of Immu- nology, 2008, 181: 2651–2663. ycobacterium tuberculosis is the causative agent of tu- unidentified components (4). In the latter study, endocytosis of a http://www.jimmunol.org/ berculosis that infects more than one-third of the mixture of lysosomal enzyme inhibitors significantly retarded M. M world’s population (1). Only 5–10% of these infected smegmatis killing, providing functional evidence that lysosomal individuals will develop tuberculosis at some point in their life (2). enzymes delivered to the phagosome contribute to this process. In A major factor in determining whether the disease is contained or contrast, pathogenic mycobacteria such as M. tuberculosis and progresses is the macrophage, which hosts mycobacteria in mem- Mycobacterium avium block phagolysosome fusion and NO re- brane-enclosed phagosomes. When macrophages are activated, lease and consequently survive and grow within phagosomes phagosomes enclosing the pathogen can fuse with lysosomes and (5). However, when the proinflammatory response of macro- the bacteria are killed. However, under steady-state conditions, the phages infected with pathogenic mycobacteria is stimulated by by guest on September 27, 2021 phagosome becomes programmed to prevent its maturation. As a some specific lipids, even pathogenic mycobacteria may be ef- consequence the bacteria survive and may grow. The mechanisms ficiently killed (6). by which lysosomal factors kill mycobacteria are poorly under- The family of NF-␬B transcription factors plays a crucial role in stood (3). In this study, we focused on the nonpathogenic Myco- the regulation of the inflammatory process (7). This family of pro- bacterium smegmatis as a model system and addressed the intra- teins is composed of two subfamilies: the NF-␬B proteins and the cellular mechanisms that are responsible for killing mycobacteria Rel proteins. NF-␬B transcription factors are present in the cyto- in mature phagosomes. plasm as heterodimers, most commonly of p65 and p50 subunits in M. smegmatis is phagocytosed by macrophages and is killed a complex with an inhibitor, I␬B (8). When proinflammatory sig- within phagosomes by a combination of factors delivered from naling occurs via activation of cell surface receptors (e.g., TLRs), lysosomes, reactive nitrogen intermediates, and likely other still the I␬B becomes phosphorylated, leading to its degradation by the proteosome system. This allows the active subunits to enter the nucleus where they up-regulate the transcription of two to three † *European Molecular Biology Laboratory, Heidelberg, Germany; Unidade de Ret- hundred genes (9). rovı´rus e Infecc¸o˜es Associadas-Centro de Patoge´nese Molecular Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; and ‡School of Biochemistry, Genetics, Mi- The products characteristic of early events of the early immune crobiology and Plant Pathology, University of KwaZulu-Natal, Pietermaritzburg, response mediated by NF-␬B include the release of ILs and in- South Africa ducible NO synthase-mediated NO production (10). However, re- Received for publication February 28, 2008. Accepted for publication May 28, 2008. active nitrogen intermediate production only plays a partial role in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance mycobacterial clearance (6, 11). It is clear that some ILs such us with 18 U.S.C. Section 1734 solely to indicate this fact. IFN-␥ and TNF-␣ increase the maturation of mycobacterial phago- 1 M.G.G. was supported by a Research Fellowship from Alexander von Humboldt somes as well as killing of mycobacteria. However, the intracel- Foundation and is currently funded by an European Molecular Biology Organization lular mechanisms by which ILs boost mycobacterial killing are Fellowship. E.A. was supported by Fundac¸a˜o para a Cieˆncia e a tecnologia Grant POCI/BIA-BCM/55327/2004 with coparticipation of the European Union fund poorly understood (12–14). FEDER Programme POCI2010. The fusion of phagosomes with late endocytic organelles (often 2 M.G.G. and B.B.M. contributed equally to this work. abbreviated as lysosomes) is usually considered to be one, perhaps 3 Address correspondence and reprint requests to Dr. Maximiliano Gabriel Gutierrez, the main effector of mycobacterial killing. This is based on exten- European Molecular Biology Laboratory, Postfach 102209, 69117 Heidelberg, Ger- sive correlative data associating killing with the late fusion events many. E-mail address: [email protected] that deliver the proton ATPase and lysosomal enzymes to phago- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 somes (15–17). For example, mutants of both M. tuberculosis and www.jimmunol.org 2652 NF-␬B CONTROLS PHAGOLYSOSOME FUSION bacillus Calmette-Gue´rin that fail to block phagosome maturation ilized water. Quantitative cultures for M. smegmatis or M. avium were are killed more effectively than wild-type bacteria (18, 19). As performed by 10-fold serial dilutions inoculated on 7H10 agar plates. Five already mentioned, a role for lysosomal enzymes in killing is sup- microliters was plated by triplicate and the number of colonies was counted after 48 h and referred as number of colonies (CFU) per milliliter. ported by our earlier studies (4). Phagocytosis and inflammation are strongly correlated (20). Indirect immunofluorescence Binding of mycobacteria to macrophage surface receptors, espe- cially TLR2 and TLR4, initiates a rapid proinflammatory response Cells were fixed with 3% paraformaldehyde solution in PBS for 10 min and which is more robust for nonpathogenic than for pathogenic my- quenched by incubating with PBS/50 mM NH4Cl. Subsequently, cells were permeabilized with 0.05% saponin in PBS containing 0.2% BSA and then cobacteria (21). This proinflammatory response is mediated by incubated with the primary and secondary Abs. Cells were mounted with MAPKs and by proinflammatory transcription factors, including Permafluor mounting medium (DAKO) and analyzed by confocal micros- NF-␬B (21). In addition, the signaling from TLR has also been copy (Zeiss LSM510). implicated in the regulation of phagosome maturation and bacterial killing (22). ELISA ␬ The data regarding the activation of NF- B by pathogenic my- The p65 activation was assayed using a multiwell assay TransAM NF-␬B cobacteria are conflicting. A number of studies have described Kit (Active Motif). Briefly, J774-infected macrophages were scraped and inhibition of NF-␬B activation by M. tuberculosis components (23, nuclear extracts were isolated. Five
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