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Macrophages Escape from Vacuoles in Monocytogenes Listeria Inducible Renitence Limits Listeria monocytogenes Escape from Vacuoles in Macrophages This information is current as Michael J. Davis, Brian Gregorka, Jason E. Gestwicki and of September 25, 2021. Joel A. Swanson J Immunol 2012; 189:4488-4495; Prepublished online 21 September 2012; doi: 10.4049/jimmunol.1103158 http://www.jimmunol.org/content/189/9/4488 Downloaded from References This article cites 52 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/189/9/4488.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 25, 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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Inducible Renitence Limits Listeria monocytogenes Escape from Vacuoles in Macrophages Michael J. Davis,* Brian Gregorka,† Jason E. Gestwicki,‡ and Joel A. Swanson*,† Membranes of endolysosomal compartments in macrophages are often damaged by physical or chemical effects of particles ingested through phagocytosis or by toxins secreted by intracellular pathogens. This study identified a novel inducible activity in macro- phages that increases resistance of phagosomes, late endosomes, and lysosomes to membrane damage. Pretreatment of murine macrophages with LPS, peptidoglycan, TNF-a, or IFN-g conferred protection against subsequent damage to intracellular mem- branes caused by photooxidative chemistries or by phagocytosis of ground silica or silica microspheres. Phagolysosome damage was partially dependent on reactive oxygen species but was independent of the phagocyte oxidase. IFN-g–stimulated macrophages from mice lacking the phagocyte oxidase inhibited escape from vacuoles by the intracellular pathogen Listeria monocytogenes, which suggested a role for this inducible renitence (resistance to pressure) in macrophage resistance to infection by pathogens that Downloaded from damage intracellular membranes. Renitence and inhibition of L. monocytogenes escape were partially attributable to heat shock protein-70. Thus, renitence is a novel, inducible activity of macrophages that maintains or restores the integrity of endolysosomal membranes. The Journal of Immunology, 2012, 189: 4488–4495. lassical macrophage activation in response to LPS, other cholesterol-dependent cytolysin, lysteriolysin O (LLO), which bacteria-derived molecules, and the cytokines IFN-g and facilitates escape from macrophage phagosomes into the cytosol. http://www.jimmunol.org/ C TNF-a is important in host defense against infections (1, Bacteria typically escape macrophage phagosomes prior to phag- 2), as well as in sepsis, cancer, and atherosclerosis (3–5). Induced osome–lysosome fusion (16). Wild-type L. monocytogenes, but not biochemical activities that counteract infection include the phago- L. monocytogenes lacking LLO (Dhly), can disrupt pH and cal- cyte NADPH oxidase (6) and inducible NO synthase (7), which cium gradients across phagosomal membranes, which delays the generate toxic reactive oxygen species (ROS) and reactive nitrogen fusion of phagosomes containing bacteria with LAMP-1–positive species, respectively. Other antimicrobial activities include increased lysosomes (16, 17). In the cytosol, L. monocytogenes multiplies phagosome–lysosome fusion, Ag presentation, iron sequestration, and eventually co-opts the host actin cytoskeleton to invade neigh- and autophagy (8–10). Bacterial infection of macrophages also boring cells. Activation of macrophages by IFN-g has a critical role increases cellular expression of heat shock protein-70 (HSP70), in protecting mice from L. monocytogenes infection (18), signif- by guest on September 25, 2021 which can be released from macrophages through nonclassical se- icantly altering the dynamics of L. monocytogenes in macrophages cretion systems (11, 12). Only a fraction of the activation-regulated (19, 20). Reactive oxygen and nitrogen species produced by ac- genes have been implicated in these processes (13), which suggests the tivated macrophages inhibit L. monocytogenes infection and es- existence of other activities that macrophages use to combat infection. cape from the macrophage phagosome (21, 22). The roles of other Classical macrophage activation was originally identified from functions of activated macrophages in prevention of L. mono- the ability of a sublethal infection with Listeria monocytogenes to cytogenes escape remain unknown. protect mice from subsequent infectious challenges (14). L. mono- In a previous study of lysosome damage, we determined that the cytogenes is a Gram-positive facultative intracellular pathogen that phagolysosomes of mouse macrophages treated with LPS were causes gastrointestinal disease or more serious infections in pregnant more resistant to damage after phagocytosis of ground silica or women, neonates, or immunocompromised individuals (15). After silica microspheres (SMS) than were phagolysosomes of unstimu- entry into cells by phagocytosis, L. monocytogenes secretes a lated macrophages (23). Earlier studies indicated roles for HSP70 in stabilizing lysosomes (24–28). As damage to endocytic compartment membranes is a necessary consequence of infections by intracellular *Graduate Program in Immunology, University of Michigan Medical School, Ann † pathogens that access host cell cytosol, increased resistance to such Arbor, MI 48109; Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; and ‡Life Sciences Institute, Uni- membrane-damaging activities would be a novel mechanism of versity of Michigan, Ann Arbor, MI 48109 resistance to infection. We therefore examined the mechanism of Received for publication November 3, 2011. Accepted for publication August 7, this novel inducible activity and the contribution of HSP70 to that 2012. resistance. We show that the protective effect could be induced by This work was supported by National Institutes of Health Grants AI 035950 (to J.A.S.) TLR ligands, IFN-g,TNF-a, intact bacteria, and exogenously added and NS 059690 (to J.E.G.). M.J.D. was supported in part by National Institutes of Health Training Grant T32 HL07749. HSP70. This activity limited damage from a variety of agents in- Address correspondence and reprint requests to Dr. Joel A. Swanson, University of cluding ground silica, SMS, light-induced membrane damage, and Michigan, Medical Sciences II Building, Room 5818, Ann Arbor, MI 48109. E-mail infection by L. monocytogenes, indicating it as a general protective address: [email protected] response of activated macrophages. Abbreviations used in this article: BMM, bone marrow-derived macrophage; DPI, diphenyleneiodonium; Fdx, fluorescein–dextran; HSP70, heat shock protein-70; LLO, lysteriolysin O; MOI, multiplicity of infection; NAC, N-acetyl-L-cysteine; PGN, pep- Materials and Methods tidoglycan; PLL, poly-L-lysine; ROS, reactive oxygen species; SMS, silica micro- Materials sphere; TRdx, Texas red–dextran; TSS, TBS plus 2% sheep serum. Fluorescein–dextran (Fdx; average molecular mass 3000 Da), RPMI 1640, Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 FCS, penicillin/streptomycin solution, valinomycin, nigericin, recombinant www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103158 The Journal of Immunology 4489 mouse IL-10, recombinant mouse IL-1b, SNARF-1 carboxylic acid acetate Photooxidative damage of lysosomes succinimidyl ester, Cell Trace CFSE Cell Proliferation Kit, Texas red– dextran (TRdx; average molecular mass 10,000 Da), Texas red–phalloidin, BMM were coloaded with 150 mg/ml Fdx along with 75 mg/ml TRdx, then and gentamicin solution were purchased from Invitrogen (Carlsbad, CA). stimulated with LPS and IFN-g for 18 h, as described earlier. TRdx was Recombinant human IFN-b, diphenyleneiodonium (DPI), and N-acetyl-L- used as a photosensitizer, and Fdx imaging was used to monitor lysosome cysteine (NAC) were purchased from Sigma Chemical Co. (St. Louis, MO). damage. BMM were rinsed with Ringer’s buffer, mounted onto the mi- LPS no. 225 Salmonella typhimurium was from List Biological Labora- croscope heated stage, and allowed to equilibrate for 5 min. A field was tories (Campbell, CA), and 35-mm dishes with attached 14-mm coverglass selected for imaging, then an initial Fdx image set was acquired. Using a were purchased from MatTek Corp (Ashland, MA). Peptidoglycan (PGN) 580-nm TRdx excitation filter, cells were then exposed to bright light in from Escherichia coli 0111:B4 was purchased from Invivogen (San Diego, regular, 5-s exposures. Between each exposure, Fdx images at 440 nm and CA). IFN-g was from R&D Systems (Minneapolis, MN), TNF-a was from 485 nm were acquired to assess lysosome damage. This process was re- 3 eBioscience (San Diego, CA), and murine IL-6 was from Calbiochem peated for 60 total seconds of
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