Inducible Renitence Limits Listeria monocytogenes Escape from Vacuoles in

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

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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 or by toxins secreted by intracellular pathogens. This study identified a novel inducible activity in macro- phages that increases resistance of , late endosomes, and 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 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 resistance to infection by pathogens that Downloaded from damage intracellular membranes. Renitence and inhibition of L. monocytogenes escape were partially attributable to heat shock -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– 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 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 –lysosome fusion, Ag presentation, iron sequestration, and eventually co-opts the host actin cytoskeleton to invade neigh- and (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 580-nm photo-exposure (12 5 s). Percent (San Diego, CA). MIN-U-SIL-15 ground silica was a gift from U.S. Fdx release was then calculated from the Fdx images for each time point. Silica (Berkeley Springs, WV). Oxide silica 3-mm microspheres (SMS; Infection of cells with L. monocytogenes Microspheres-Nanospheres, Cold Spring, NY) were washed overnight in 1 N HCl then rinsed several times in distilled water. For some experiments, Wild-type L. monocytogenes strain (DP-L10403) and Dhly L. mono- SMS were coated with 0.1 M poly-L-lysine (PLL) for 30 min. Recombi- cytogenes (DP-L2161) were provided by Daniel Portnoy (University of nant HSP70 and HSC70 were purified as in Ref. 29. The synthesis of Ym1 California, Berkeley). L. monocytogenes cultures were grown overnight at (also known as MKT077) has been described (30). room temperature then diluted and grown 75 min at 37˚C with shaking to Bone marrow-derived macrophages OD 600 nm = 0.8. One milliliter of bacterial culture was then centrifuged (2 min at 5000 3 g) and resuspended in Ringer’s buffer. Bacteria were C57BL/6J (wild type), B6.129S-Cybbtm1Din/J (nox2 deficient), and TLR42/2 stained using SNARF-1 or CFSE dye for 15 min in a shaking 37˚C in- mice (strain C57BL6/10SCN) were purchased from The Jackson Laboratory cubator. The culture was then washed three times in Ringer’s buffer. BMM Downloaded from (Bar Harbor, ME). All mice were maintained under specific pathogen-free were infected by adding bacteria to 200 ml of culture medium without conditions at the University of Michigan animal facility. Differentiation of antibiotics and replacing the BMM culture medium with this infection mix. macrophages from mouse bone marrow cells has been previously described All infections were 10 min followed by rinsing in Ringer’s buffer and (23, 31). Briefly, marrow cells extracted from mouse femurs were cultured chasing in culture medium containing gentamicin. After 90–120 min of for 6 d in the presence of M-CSF, which was provided by 30% L-cell- culture, infected cells were either imaged for damage or fixed to measure conditioned medium. Bone marrow-derived macrophages (BMM) were L. monocytogenes escape.

frozen and stored at 2130˚C as aliquots in M-CSF–containing medium http://www.jimmunol.org/ with 10% DMSO and thawed as needed for experiments. Measurement of vacuole escape by L. monocytogenes Loading macrophage lysosomes and stimulation of cells BMM infected with CFSE-stained L. monocytogenes were fixed using cytoskeleton fix (2% paraformaldehyde, 30 mM HEPES, 10 mM EGTA, After allowing BMM to attach for several hours in RPMI 1640 with 10% 0.5 mM EDTA, 5 mM MgSO4, 33 mM potassium acetate, 5% polyethylene FCS and penicillin/streptomycin, cells were incubated overnight in medium glycol 400) and permeabilized in 0.1% Triton X-100 in PBS. Coverslips containing 150 mg/ml Fdx. BMM were then rinsed and chased in unlabeled were blocked in PBS containing 2% goat serum and stained using Texas medium for at least 3 h to allow Fdx trafficking into late compartments of red–phalloidin and DAPI. Cells were imaged and the numbers of total the endolysosomal network (32). Penicillin/streptomycin was omitted from (DAPI-positive) and escaped (Texas red–phalloidin–positive) bacteria was BMM cultures to be infected with L. monocytogenes. LPS (100 ng/ml), recorded for each infected macrophage. PGN (2.5 mg/ml), IFN-g (100 U/ml), TNF-a (100 ng/ml), IL-6 (5 ng/ml), by guest on September 25, 2021 IL-10 (100 ng/ml), IL-1b (10 ng/ml), or IFN-b (100 U/ml) were included Immunofluorescence microscopy in both the Fdx pulse medium and the subsequent Fdx-free chase medium. BMM on 12-mm circular coverslips were stimulated overnight with IFN-g Uncoated SMS were fed to BMM in serum-free medium or PLL-coated or LPS, then prepared for immunofluorescence microscopy. Cells were SMS were fed to cells in medium containing 10% FCS and incubated 1 h fixed for 60 min at 37˚C (fixative: 2% paraformaldehyde, 4.5% sucrose, before imaging. 20 mM HEPES, pH 7.4, 70 mM NaCl, 10 mM KCl, 10 mM MgCl2,2mM Measurement of damage to endolysosomal compartments EGTA, 70 mM lysine-HCl, 10 mM sodium periodate), rinsed (TBS: 20 mM Tris-HCl, pH 7.4, 4.5% sucrose, 150 mM NaCl), extracted with Endolysosomal membrane damage was measured in live BMM by epi- 220˚C methanol, rinsed with TBS plus 2% sheep serum (TSS), incubated fluorescence ratiometric microscopy, as described previously (23). Cells with primary Abs against HSP70 (rabbit anti-HSP70/HSC70; Santa Cruz loaded with Fdx were rinsed and imaged in Ringer’s buffer (155 mM Biotechnology) and LAMP-1 (rat monoclonal 1D4B; Developmental Studies NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4,10mM Hybridoma Bank, Iowa City, IA) in TSS, rinsed with TSS, incubated with HEPES, and 10 mM ) using a Nikon TE300 inverted microscope secondary Abs (Alexa Fluor 488-labeled goat anti-rat IgG; Alexa Fluor equipped with a mercury arc lamp, 360 plan-apochromat 1.4-numerical 594-labeled goat anti-rabbit IgG; Invitrogen), rinsed, mounted in anti-fade, aperture objective, a cooled digital CCD camera (Quantix Photometrics, and observed by digital fluorescence microscopy. The fluorescence of Tucson, AZ), a temperature-controlled stage, and a Fura/FITC ratiometric Alexa 488-labeled HSP70 was quantified from digital images of 10 cells dichroic mirror (Omega Optical, Brattleboro, VT). Three images were for each condition. acquired for each field of cells: one phase-contrast image and two fluo- rescence images, which used a 510-nm emission filter and two different Statistical methods excitation band-pass filters (centered at 440 nm and 485 nm), mounted in Student t test was performed on groups of cells containing similar amounts computer-controlled filter wheels (Sutter Instruments, Novato, CA). Met- of SMS, L. monocytogenes, or photostimulation. amorph software (Molecular Devices, Downingtown, PA) was used for image capture and analysis. The Fdx signal in the 440-nm excitation channel is relatively insensitive Results to pH, whereas the signal in the 485-nm excitation channel varies between Macrophage activation stabilizes lysosomes pH 4.5 and 7.5. Thus, the ratio of Fdx fluorescence in the 485-nm channel divided by that in the 440-nm channel is related to pH in any subregion of Previous studies established that particle-mediated lysosome an image (pixel). For calibration, 485-nm/440-nm excitation ratios were damage was reduced after macrophages were stimulated with LPS measured in cells with intracellular pH fixed at pH 9, 7.5, 7.0, 6.5, 6.0, 5.5, (23). To determine which other agents could induce this effect, 5.0, 4.5, and 4.0, using ionophores nigericin and valinomycin (10 mM) in BMM late endosomes and lysosomes were loaded with Fdx and clamping buffers (130 mM KCl, 1 mM MgCl2, 15 mM HEPES, 15 mM MES). Ratios were converted to pH, as described previously (17, 23, 33). stimulated with cytokines or TLR ligands, then the damage in- A four-variable sigmoidal standard curve was then constructed using duced by phagocytosis of SMS was measured as the release of Fdx Sigmaplot software (San Jose, CA). Regions showing pH greater than pH from endolysosomal compartments into (Fig. 1A, 1B). 5.5 were taken to indicate lysosome release. The percent of Fdx released in cells was calculated by summing the 440-nm intensity of pixels in regions LPS, PGN, IFN-g, and TNF-a protected endolysosomes from of pH above 5.5 and dividing this value by the summed 440-nm intensity SMS challenge, whereas IL-6, IL-10, IL-1b, and IFN-b did not. for the entire cell. Time-course measurements indicated that full lysosome protection 4490 INDUCIBLE LYSOSOME RENITENCE IN MACROPHAGES required 18 h of LPS stimulation, although significant protection Macrophages were pretreated with DPI, which is a potent inhibitor was observed after 5 h (Fig. 1C). We termed this inducible membrane of ROS-generating activities in macrophages, including the phago- resistance or repair mechanism renitence: resistance to pressure. cyte oxidase (NOX2) and the mitochondrial electron transport chain The studies of MacKaness (14), which first described a role for (39–41). Macrophages loaded with Fdx and incubated in DPI or activated macrophages in host defense, showed that infection of control medium were fed ground silica or SMS, and the extent of mice with sublethal doses of L. monocytogenes led to differenti- damage was assessed. DPI reduced phagolysosome damage after ation of macrophages with increased resistance to subsequent phagocytosis of both kinds of particle (Fig. 2A, 2B). Similarly, L. monocytogenes infections. Although activation was eventually BMM preincubated in the antioxidant NAC showed less SMS- attributed to the action of IFN-g and TNF-a (34), we postulated mediated lysosome damage than that of control cells (Fig. 2C). that some of the increased resistance may be induced directly by This reduced damage was independent of any DPI effect on the bacteria. We therefore measured the extent to which phagocytosis efficiency of phagocytosis, as DPI-treated BMM that had phago- of hemolysin-deficient (Dhly) L. monocytogenes, which do not cytosed SMS showed less lysosome damage than control cells that damage vacuoles (17, 35, 36), increased macrophage resistance to had ingested equivalent numbers of particles. These data indicate subsequent challenge by phagocytosed SMS. BMM that ingested that phagocyte oxidase-independent ROS contribute to silica- Dhly L. monocytogenes became resistant to lysosome damage induced lysosome damage. Renitence may therefore consist of after phagocytosis of SMS (Fig. 1D). The subset of BMM that mechanisms that interfere with ROS generation in cytoplasm. were exposed to Dhly L. monocytogenes but which did not ingest A major source of ROS in the phagosomal lumen is the phagocyte bacteria showed lysosome damage levels comparable to unsti- oxidase. We examined the contribution of phagocyte oxidase- mulated cells (Fig. 1E), indicating that the Dhly L. monocytogenes generated ROS to damage by comparing wild-type and phagocyte Downloaded from activated this protective mechanism inside the cells that ingested oxidase (nox2)-deficient BMM and observed similar levels of phag- them, rather than through some secreted factor. This suggests that olysosome damage after phagocytosis of ground silica (Fig. 2D) or microbes ingested by macrophages initiate a cytokine-independent SMS (Fig. 2E). This indicates that the phagocyte oxidase does not differentiation to a more resistant phenotype. contribute to particle-mediated lysosome damage. ROS contribute to particle-mediated lysosome damage L. monocytogenes hemolysin damages endolysosomes http://www.jimmunol.org/ Inducible renitence could interfere with the mechanism(s) by The relationship between L. monocytogenes vacuolar escape and which particles damage membranes. As ROS have been implicated endolysosome damage was investigated. A previous study suggested in NLRP3 inflammasome activation by lysosome damage (37, 38), that at a low multiplicity of infection (MOI), L. monocytogenes we examined the contribution of ROS to phagolysosome damage. escapes from LAMP-1–negative, late endosome-like vacuoles (16), by guest on September 25, 2021

FIGURE 1. Macrophage activation increases lysosome renitence. (A and B) BMM were loaded with Fdx, chased for 3 h in medium without Fdx, then fed SMS and imaged to measure lysosome damage. The number of SMS phagocytosed by each BMM was counted, and groups of BMM with similar numbers of SMS phagosomes were compared. LPS, PGN, IFN-g, and TNF-a increased macrophage resistance to damage by SMS; IL-6, IL-10, IL-1b, and IFN-b were not protective. Bars and data points are the average percent of Fdx released (6SEM, n $ 100 cells). (C) Time course of induction by LPS. LPS was included in the medium for the indicated times. BMM stimulated for 18 h showed significantly less damage than those stimulated for 0, 1, or 2 h (p , 0.001) or 6 h (p , 0.05); n . 30 cells. (D) BMM were fed SNARF-1–stained, hly-deficient (Dhly) L. monocytogenes or stimulated with LPS, then fed 17 h later with SMS to measure damage (n . 200 cells). Damage was reduced by LPS or by prior infection with Dhly L. monocytogenes.(E) Replotted data from the Dhly-induced, SMS-fed condition in (D). Using the SNARF-1 fluorescence, BMM were separated into cells that had phagocytosed at least one bac- terium (Dhly; dark bar) and those cells in the same coverslips that had not ingested bacteria (w/o Dhly; striped bar). Bars in (A)–(C) represent cells that contained seven to nine SMS. Bars in (D) and (E) represent cells containing one to three SMS. *p , 0.05, **p , 0.001. The Journal of Immunology 4491

FIGURE 2. Nonphagosomal reactive oxygen intermediates contribute to silica-mediated lysosome damage. BMM were loaded with Fdx then fed ground silica (A, D) or PLL-coated SMS (B, C, E). Then, 5 mM DPI (A, B)or50mMNAC(C) was added to the medium with the particles. (D and E) BMM from wild-type or nox2-deficient (Nox22/2) mice were loaded with Fdx, fed particles, and scored for lysosome damage. All data points represent mean 6 SEM Fdx release with n . 200 BMM for (A), (B), (D), and (E) and n . 50 BMM for (C). *p , 0.05, **p , 0.001.

which suggests that escape could occur without causing release of confirming that both activities required LLO. Thus, although at Downloaded from Fdx from late endocytic compartments. To determine if infection low MOI L. monocytogenes entered cytoplasm with negligible at higher MOI damages late endocytic compartments, Fdx-loaded damage to late endocytic compartments, higher MOI resulted in LLO- BMM were fed SNARF-1–labeled wild-type or Dhly L. mono- dependent release of Fdx from those compartments into cytoplasm. cytogenes and assayed for endolysosome damage. In cells con- taining fewer than seven bacteria, wild-type L. monocytogenes Inducible renitence inhibits L. monocytogenes escape and associated lysosome damage

showed low levels of lysosome damage, consistent with a mech- http://www.jimmunol.org/ anism in which L. monocytogenes escapes from vacuoles at earlier The phagocyte oxidase inhibits L. monocytogenes escape in acti- stages of maturation. However, higher bacterial loads showed vated macrophages (21, 22). To determine if inducible renitence more damage, which correlated with the number of L. monocytogenes also contributes to the inhibition of L. monocytogenes escape in per BMM (Fig. 3A–D). BMM infected with Dhly L. monocytogenes activated macrophages, wild-type and nox2-deficient BMM were showed neither lysosome damage nor L. monocytogenes escape, loaded with Fdx, activated with IFN-g, infected with wild-type by guest on September 25, 2021

FIGURE 3. Inducible renitence protects IFN-g–activated macrophages from L. monocytogenes escape and damage. (A–C) Damage by infection with L. monocytogenes. C57/BL6 BMM were loaded with Fdx overnight, chased in dye and antibiotic-free medium for at least 3 h, infected with SNARF-1–labeled wild-type or Dhly L. monocytogenes, chased in gentamicin-containing medium for 90 min, and imaged to measure lysosome damage. (A) Fdx pH map of a BMM infected with wild-type L. monocytogenes. Blue pixels indicate lysosome damage (i.e., cytoplasmic Fdx). (B) Fdx pH map of a BMM infected with Dhly L. monocytogenes indicates that the lysosomes are intact. Scale bar, 10 mm. Green pixels in (A)and(B) indicate SNARF-1–labeled L. monocytogenes.(C) Average 6 SEM percent Fdx release for BMM (n . 100 for each group) plotted in groups of cells containing similar numbers of total wild-type (filled bars) or Dhly (open bars) L. monocytogenes.(D) Fdx release data replotted from (C) showing average number of escaped (phalloidin-positive) L. monocytogenes 6 SEM (n . 100 for each group) plotted on the x-axis for each category of BMM grouped by total number of L. monocytogenes per cell (in parentheses). Filled circles represent groups of BMM infected with wild-type L. monocytogenes; open symbol near the origin represents all groups of Dhly L. monocytogenes.(E and F) C57/BL6 (WT) and nox2-deficient (Nox22/2) BMM were stimulated with IFN-g (open bars) or left unstimulated (dark bars) and loaded with Fdx. BMM infected 120 min with wild-type L. monocytogenes were fixed and stained with phalloidin to measure L. monocytogenes escape (E) or imaged for Fdx release (F). Bars in (E) are average percent of phalloidin-positive L. monocytogenes and error bars are SEM (n $ 350 cells). Bars in (F) indicate average percent of Fdx released 6 SEM of BMM containing one to seven L. monocytogenes per cell (n $ 130 cells). *p , 0.05, **p , 0.001. L.m., L. monocytogenes. 4492 INDUCIBLE LYSOSOME RENITENCE IN MACROPHAGES

L. monocytogenes, and scored for actin-positive cytoplasmic bac- lysosomal resistance to photodamage (24), we hypothesized that teria. IFN-g treatment limited L. monocytogenes escape in both HSP70 contributed to induced renitence. Macrophages were incu- wild-type and nox2-deficient BMM. Although IFN-g–activated, bated with recombinant HSP70 or LPS, and endolysosome damage nox2-deficient BMM did not inhibit escape as efficiently as was measured after SMS phagocytosis. Exogenous HSP70 reduced IFN-g–activated wild-type BMM, they were significantly better lysosome damage to levels comparable to those in LPS-activated than unactivated, nox2-deficient BMM (Fig. 3E). IFN-g–activated, cells (Fig. 5A). To rule out endotoxin contamination as an alter- nox2-deficient BMM also showed reduced levels of endolysosome native explanation for HSP70-mediated lysosome protection (43), damage after L. monocytogenes infection compared with unac- damage was measured in TLR4-deficient BMM. These cells showed tivated nox2-deficient cells (Fig. 3F). This nox2-independent resis- significant resistance to damage when treated with recombinant tance suggests that inducible renitence inhibits L. monocytogenes HSP70 or IFN-g but not with LPS (Fig. 5B, 5C). HSP70-mediated escape from vacuoles of activated macrophages. However, con- protection was also inhibited by the inhibitors of HSP70 ATPase tributions of other IFN-g–inducible activities to macrophage re- activity Ym1 (30, 44) and Myr (29, 45, 46) (Fig. 5B). HSC70 in- sistance to L. monocytogenes escape cannot be excluded. duced less protection than HSP70 (Fig. 5C), consistent with pre- Inducible renitence protects lysosomes against photooxidative vious observations indicating the specificity of HSP70 for lysosome- damage protective effects (24). To test the hypothesis that endogenous HSP70 contributes to inducible lysosome renitence, macrophages were Inducible renitence was also measured in a nonparticulate endo- loaded with Fdx and stimulated with LPS for 0, 5, or 18 h. For lysosome-damaging assay. Late endosomes and lysosomes were the last 5 h before phagocytosis of SMS, cells were treated with loaded by endocytosis with TRdx, as a photosensitizer, and Fdx, to Downloaded from HSP70 inhibitors Ym1 and Myr. Macrophages stimulated with monitor membrane integrity. Cells were stimulated with LPS or LPS in the presence of inhibitors showed greater endolysosome IFN-g, then exposed to 580-nm light, which excites TRdx but not Fdx. The excited TRdx increases photooxidative chemistries that damage than cells without drug (Fig. 5D), indicating that HSP70 damage cell membranes (24, 42). Lysosome integrity was mea- mediates LPS-induced renitence. The contribution of HSP70 to in- sured by imaging Fdx after each 580-nm exposure. In unstimu- ducible renitence was also tested in an L. monocytogenes infection lated BMM, lysosome damage was detectable after 25-s exposure model. TLR4-deficient BMM were treated with HSP70 or IFN-g, http://www.jimmunol.org/ and was nearly complete by 50 s (Fig. 4). Control BMM, in which infected with L. monocytogenes, and scored for escape. HSP70- endolysosomes were loaded with Fdx but not TRdx and exposed treated BMM showed a reduction in L. monocytogenes escape similarly to 580-nm light, showed no damage. Half-maximal ly- comparable to that of IFN-g–stimulated cells (Fig. 5E). Inhibitors sosome damage in LPS-stimulated or IFN-g–stimulated BMM re- of HSP70 partially reversed the IFN-g–mediated reduction in quired greater exposure to light than in unstimulated BMM (Fig. 4), L. monocytogenes vacuolar escape, indicating that HSP70-mediated Thus, the inducible resistance to membrane damage also affects membrane protection inhibits L. monocytogenes vacuolar escape nonphagosomal lysosomes. and that HSP70 contributes to inducible renitence. Immunofluorescence microscopy indicated that total levels of HSP70 limits damage

HSP70 increased in BMM stimulated with LPS (Fig. 5F, 5G) and by guest on September 25, 2021 As infection can increase synthesis and secretion of HSP70 from IFN-g (data not shown) and that HSP70 localized to small, LAMP-1– macrophages (11, 12) and exogenously added HSP70 can increase negative vesicles. Lack of colocalization between HSP70 and

FIGURE 4. Inducible renitence protects against photodamage. BMM lysosomes were loaded with Fdx and TRdx. Cells were exposed to 580-nm light from the microscope arc lamp in 5-s pulses; Fdx pH images were acquired between each pulse. (A) Sample pH maps of fields of photostimulated BMM, with pH color key on the right and a 50-mm scale bar below the images (right side). (B) Percent of Fdx released was calculated for each cell. Average percent release for each time point is plotted 6 SEM. At least 50 cells were analyzed for each condition. Control macrophages were significantly different from both LPS- stimulated and IFN-g–simulated cells with p , 0.001 for all data between 30 to 50 s and p , 0.05 for the data at 25 and 55 s (unpaired Student t test). The Journal of Immunology 4493 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. HSP70 contributes to inducible renitence. (A–D) HSP70 inhibits damage by SMS. (A) Fdx-loaded BMM were exposed to LPS or 300 nM recombinant HSP70 for 5 h, then cells were rinsed and fed SMS for 1 h and imaged for Fdx release (1–3 SMS/cell, n . 100 cells). (B) BMM from TLR42/2 mice were exposed to IFN-g overnight, LPS for 5 h, or 300 nM HSP70 for 5 h, with or without HSP70 inhibitors Ym1 (20 mM) or Myr (25 mM). HSP70 inhibited lysosome damage (n . 300 cells). (C) TLR42/2 BMM were exposed to IFN-g overnight or 300 nM recombinant HSP70 or HSC70 for 5 h, then assayed for SMS damage (n . 180 cells). (D) Wild-type BMM were exposed to LPS for 5 or 18 h or left untreated. Ym1 (20 mM) or Myr (25 mM) were included for the last 5 h of stimulation (n . 120 cells). (E) TLR42/2 BMM were exposed to recombinant HSP70 for 5 h or to IFN-g overnight. Indicated IFN-g–stimulated cells were also incubated with Ym1 or Myr for the last 5 h of stimulation. BMM were infected with wt or Dhly L. monocytogenes, and vacuolar escape was measured (n . 500 cells). Bars are population averages (6 SEM). *p , 0.05, **p , 0.001. (F and G) Immunofluorescence localization of HSP70 and LAMP-1 in (F) unstimulated BMM and (G) IFN-g–stimulated BMM. Overlay images show corresponding distributions of LAMP-1 (green) and Hsp70 (red). Scale bar, 10 mm.

LAMP-1 suggests that HSP70 effects on renitence do not occur microsphere silica particles, LLO-dependent membrane perfora- from within the endolysosomal network. tion, and photooxidative chemistries. Thus, inducible renitence is unlikely to involve mechanisms specific to any one kind of Discussion damage but instead represents a set of activities that reinforce This study identifies inducible renitence as a novel common feature endolysosomal membranes. Although the assays used here allowed of classical macrophage activation, which can be induced by the us to analyze renitence as a feature of late endosomes and lyso- TLR ligands LPS and PGN, by whole bacteria, and by the cytokines somes, it is possible that other membranous compartments, such as IFN-g and TNF-a. Renitence was observed in a variety of different early endosomes or endoplasmic reticulum, may exhibit renitence, membrane-damaging insults, including phagocytosis of ground or as well. It will be interesting to determine if renitence is inducible 4494 INDUCIBLE LYSOSOME RENITENCE IN MACROPHAGES in other common targets of infection, such as dendritic cells and sphingomyelinase, which, in turn, catalyzes phospholipid chem- epithelial cells. istries that increase damage-resistance of lysosomal membranes in Renitence was induced by mediators of classical activation in fibroblasts (24). The evidence presented in this study indicates that macrophages, indicating a role in inflammation and host defense activated macrophages induce a similar state in their lysosomes against infections. Previous studies showed that resistance of (Fig. 5). HSP70 levels were increased by LPS and IFN-g,but lysosomes to various kinds of damage can be induced by LPS (23), HSP70 did not localize to lysosomes. This suggests that the effect TNF-a (47), or HSP70 (24), all of which are associated with in- of HSP70 on renitence may be autocrine. As HSP70 inhibitors fection. That infection by Dhly L. monocytogenes could also in- only partially reversed lysosome renitence induced by LPS (Fig. duce renitence suggests that early signaling by TLRs induces 5D) or IFN-g (Fig. 5E), we hypothesize that HSP70-mediated renitence as a protection against subsequent infections. Local in- protection is one of several membrane-stabilizing activities that duction by Dhly L. monocytogenes (Fig. 1E) suggests that at least compose inducible renitence. some of the induction is cell autonomous. It remains to be de- Thus, renitence is a novel pathway induced in activated mac- termined if renitence is inducible by TLR signaling, by membrane rophages that reduces damage to endolysosomal membranes caused damage itself, or by bacterial HSP70 (i.e., DnaK). by mechanical forces or by L. monocytogenes. This suggests that In cells containing low numbers of bacteria, L. monocytogenes therapeutic interventions that increase renitence could be effica- escape occurred with negligible damage to late endosomes and cious against infection by intracellular pathogens or in reducing lysosomes (Fig. 3D). As lysosomal perforation can activate inflam- inflammation due to microparticles or nanoparticles. masomes (38), the escape of L. monocytogenes from prelysosomal compartments may function as an immune evasion strategy. Al- Acknowledgments Downloaded from though macrophages containing few bacteria showed little dam- We thank Yoshinari Miyata for synthesis of Ym1. age, cells containing many bacteria showed extensive endolysosomal damage, which suggests that the mechanisms that allow L. mono- Disclosures cytogenes to escape prior to phagosome–lysosome fusion may break The authors have no financial conflicts of interest. down later in infection when more bacteria are present in an area.

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