Oxidase Macrophages Lacking a Functional NADPH Granulomatous Disease Monocytes and in Chronic Granulibacter Bethesdensis Persist

Persistence of the Bacterial Pathogen Granulibacter bethesdensis in Chronic Granulomatous Disease Monocytes and Macrophages Lacking a Functional NADPH This information is current as Oxidase of September 26, 2021. Jessica Chu, Helen H. Song, Kol A. Zarember, Teresa A. Mills and John I. Gallin J Immunol 2013; 191:3297-3307; Prepublished online 16 August 2013; Downloaded from doi: 10.4049/jimmunol.1300200 http://www.jimmunol.org/content/191/6/3297 http://www.jimmunol.org/ References This article cites 77 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/191/6/3297.full#ref-list-1

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Persistence of the Bacterial Pathogen Granulibacter bethesdensis in Chronic Granulomatous Disease Monocytes and Macrophages Lacking a Functional NADPH Oxidase

Jessica Chu, Helen H. Song, Kol A. Zarember, Teresa A. Mills, and John I. Gallin

Granulibacter bethesdensis is a Gram-negative pathogen in patients with chronic granulomatous disease (CGD), a deﬁciency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to nonoxidative killing mechanisms. Although G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human mono- Downloaded from cytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with IFN-g to maintain this killing effect. Although in vitro IFN-g did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN- g therapy. In IFN-g–treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 d) without decreasing viability http://www.jimmunol.org/ of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, most likely relating to the persistence of this microorganism in infected CGD patients. The Journal of Immunology, 2013, 191: 3297–3307.

ranulibacter bethesdensis, a member of the Acetobac- phonuclear leukocytes (PMN) in a complement-dependent manner teraceae family, is a human pathogen that causes in- (13). CGD PMN were incapable of killing G. bethesdensis,sug- G fection in patients with chronic granulomatous disease gesting that this organism is resistant to nonoxidative killing (CGD), a deﬁciency in the NADPH oxidase (1, 2). This Gram- mechanisms. PMN are short-lived cells that undergo apoptosis and negative, rod-shaped bacterium has been isolated from at least efferocytosis by macrophages for inﬂammation resolution (14). by guest on September 26, 2021 six CGD patients in the United States and Spain (3–5). G. be- Delayed PMN apoptosis and defective macrophage efferocytosis thesdensis can cause recurrent infections in a CGD patient over are observed in CGD (15, 16). Given that G. bethesdensis persists several years that may be the result of reinfection with different for 24 h in CGD PMN, in contrast to ∼50% clearance of the or- bacterial strains or reactivation of the same strain (3, 4). This ganism by normal PMN in the same time frame (13), we hy- organism displays a high degree of resistance to host defenses, pothesized that long-term intracellular bacterial survival in the as evidenced by isolation from spleens of wild-type mice 76 d host might rely on longer-lived macrophages and precursor mono- postinfection and from excised lymph nodes of a CGD patient cytes. In this study, we show that normal monocytes and monocyte- 5 mo after the onset of symptoms and unresponsiveness to anti- derived macrophages (MDM) internalize G. bethesdensis and kill microbial treatment (3). The increasing numbers of clinical in- it in a NADPH oxidase-dependent manner, whereas this organism fections caused by G. bethesdensis as well as other species of persists and proliferates in CGD monocytes and MDM. Resistance acetic acid bacteria (6–12) underscore the need for a better un- of G. bethesdensis to NADPH oxidase-independent myeloid derstanding of the microbial pathogenesis of the Acetobacteraceae host defenses most likely plays an important role in long-term family. infections observed in CGD patients. Previous work has demonstrated that G. bethesdensis is serum resistant and internalized by human peripheral blood polymor- Materials and Methods Ethics statement and cell isolation

Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Blood samples were obtained after informed consent from healthy and CGD National Institutes of Health, Bethesda, MD 20892 donors enrolled at the National Institutes of Health Clinical Center, as Received for publication January 22, 2013. Accepted for publication July 11, 2013. defined in National Institutes of Health institutional review board–approved protocols. Human PMN were isolated from citrated peripheral blood, as This work was supported by the Intramural Research Program of the National Insti- previously described (17). For the monocyte isolation, every 10 ml citrated tute of Allergy and Infectious Diseases at the National Institutes of Health. peripheral blood was diluted 1:3 in HBSS2 and layered on 15 ml 53% The views expressed here are those of the authors and not necessarily those of the Percoll (diluted in PBS without Ca2+,Mg2+). PBMC layers were collected, U.S. Government. and cells were washed twice in HBSS2. Monocytes were isolated with Address correspondence and reprint requests to Dr. John I. Gallin, National Institute CD14 Ab-coupled magnetic beads, according to the manufacturer’s in- of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room structions (Miltenyi Biotec, Auburn, CA). Monocyte purity (∼85–95%) 6-2551, Bethesda, MD 20892. Email address: [email protected] was confirmed using CD14 surface staining and flow cytometry as well as + Abbreviations used in this article: CGD, chronic granulomatous disease; MDM, differential staining and light microscopy. In some cases, CD14 cells were monocyte-derived macrophage; MOI, multiplicity of infection; PMN, polymorpho- further differentiated into MDM by culturing in RPMI 1640 containing nuclear leukocyte; PRR, pattern recognition receptor; ROS, reactive oxygen species. 2mML-glutamine, 10 mM HEPES (pH 7.2), 10% autologous serum, and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300200 3298 G. BETHESDENSIS PERSISTENCE IN CGD MONOCYTES/MACROPHAGES

50 ng/ml M-CSF (Invitrogen, Carlsbad, CA) for 7 d. Media were replaced a Bio-plex Pro 17-plex Human Cytokine Assay Kit analyzed on a Bio-plex every 3–4 d. Where indicated, monocytes and MDM were treated with system 200 running Bio-plex Manager (version 5.0), according to the 65 U IFN-g/ml (eBioscience, San Diego, CA) or PBS control for 2 d. Serum manufacturer’s instructions (Bio-Rad, Hercules, CA). was collected from BD serum separator tubes, according to the manufacturer’s instructions, and used fresh or snap frozen and stored at 280˚C. XTT viability assay Freshly isolated monocytes (2 3 105/200 ml/well) were infected with G. Bacterial strains and cultures bethesdensis at the indicated MOIs in 96-well plates (precoated with 10% G. bethesdensis NIH1.1 was cultured for 2 d and subcultured for 1 d to autologous serum, as described above) in the presence of autologous serum midlog phase in YPG medium (13). Escherichia coli TOP10 was grown for for 24 h before addition of 50 ml XTT reagent (1 mg/ml XTT, 25 mM 1 d and subcultured for 3 h to midlog phase in Lysogeny broth. Washed phenazine methosulfate). After 4 h, the absorbance was measured at 450 bacteria were enumerated by OD 600 nm using a NanoDrop ND-1000 nm with a Beckman Coulter DTX 880 Multimode Detector, and the (Grace Scientiﬁc, Clarksburg, MD). background OD of media alone was subtracted to give corrected OD.

Bacterial internalization assays Data analyses Cytospins. Freshly isolated monocytes (2.5 3 105/condition) were infected Flow cytometry was performed on a BD FACS Canto, and the data were with G. bethesdensis for 1 h at the indicated multiplicity of infections analyzed using FlowJo (Tree Star, Ashland, OR). All other data were (MOIs) in 96-well plates. Plates had been precoated with 10% autologous graphed and analyzed statistically using GraphPad Prism (version 5; serum for at least 1 h at 37˚C and washed three times with HBSS2. Cells GraphPad Software, La Jolla, CA). For statistical tests, *p # 0.05, **p # were incubated with no serum, autologous serum, or autologous serum 0.01, ***p # 0.001, ****p # 0.0001. incubated for 30 min at 56˚C, which inactivates complement. Heat- inactivated serum was centrifuged at 10,000 3 g for 10 min at 4˚C to pellet protein aggregates. Plates were centrifuged at 362 3 g for 8 min at Results Downloaded from 4˚C to synchronize phagocytosis. Cells were removed with Cellstripper Internalization of opsonized G. bethesdensis by CD14+ (Mediatech, Manassas, VA), subjected to cytospin, and differentially stained monocytes with HARLECO Hemacolor stain set (EMD Chemicals, Billerica, MA). Each sample was imaged, and intracellular bacteria were scored in a blinded Serum contains opsonins, including heat-stable Abs and heat-labile manner. complement proteins, which coat microbes and can trigger their Confocal and epifluorescence microscopy. A total of 3 3 105 or 5 3 105 uptake by phagocytes. We tested whether normal CD14+ mono- freshly isolated monocytes was adhered to 12- or 18-mm ethanol-washed cytes internalize G. bethesdensis and whether this process was http://www.jimmunol.org/ G. bethesdensis cover glasses in 24- or 12-well plates, respectively. was serum dependent. Most bacteria were internalized in the presence labeled with pHrodo Red succinimidyl ester (Invitrogen), according to the manufacturer’s instructions. pHrodo-labeled bacteria were incubated with of serum after 1 h at MOIs of 10:1 (Fig. 1A) and 20:1 (data not cells at the indicated MOIs and time points in the presence of serum or shown), as assessed by light microscopy. Little internalization heat-treated serum (as described above) in RPMI 1640 containing 25 mM occurred in the absence of serum, and there was ∼50% reduction 3 HEPES (pH 7.2). Plates were centrifuged at 362 g for 8 min at 4˚C to in internalization by normal monocytes incubated with heat-treated synchronize phagocytosis. Cells were washed with cold PBS twice, fixed with 4% paraformaldehyde for 5 min at room temperature in the dark, and serum. Confocal microscopy of normal monocytes incubated with washed with cold PBS or HBSS twice. Cover glasses were mounted on G. bethesdensis labeled with pHrodo, a dye that fluoresces more glass slides using VECTASHIELD Mounting Medium with DAPI (Vector intensely at an acidic pH, also demonstrated greater internalization Laboratories, Burlingame, CA), or 1 mg/ml DAPI was added to the last of bacteria in the presence of serum, but not heat-treated serum by guest on September 26, 2021 wash prior to mounting, and imaged using a Leica SP5 X-WLL confocal (Fig. 1B). CGD monocytes internalized a similar amount of microscope or a Leica Episcope with a DFC360FX camera (Leica Micro- systems). For kinetic analyses, at least 100 cells were scored for the pres- bacteria as normal monocytes in the presence of fresh serum ence of internalized pHrodo bacteria using Imaris software (Bitplane, (p = 0.114, Mann–Whitney U test) and less so in the presence of South Windsor, CT). heat-treated serum (p = 0.081, Mann–Whitney U test) (Fig. 1A). CGD monocytes followed a similar time course of pHrodo- Luminol-enhanced chemiluminescence bacteria internalization as normal monocytes, with maximal up- Bacterial activation of PMN or monocyte luminol-enhanced chemilumi- take by 30 min postinfection (Fig. 1C). Maximal internalization at nescence was measured at the indicated MOIs in an assay composed of 30 min was also confirmed by scoring the number of pHrodo- PMN or monocytes at 1 3 106/ml in RPMI 1640 medium with 10% autologous serum, 25 mM HEPES (pH 7.2), and 50 mM luminol, essentially labeled bacteria per cell (data not shown). Little to no internali- as previously described (13). zation occurred in the absence of serum for either cell type (data not shown). These results show that monocytes internalize Bacterial killing assays G. bethesdensis, and this is partially dependent on a temperature- Freshly isolated PMN, freshly isolated monocytes, or 2-d-old monocytes or sensitive serum component for normal monocytes. MDM treated with 65 U IFN-g/ml or PBS control were infected with G. + bethesdensis (preopsonized with serum) in 96-well plates for the indicated G. bethesdensis activation of the NADPH oxidase in CD14 times and at the indicated MOIs. Plates were precoated with 10% autol- monocytes ogous serum, as described above. At each time point, a final concentration of 0.5% saponin was added per well. Plates were incubated on ice for 10 The phagocyte NADPH oxidase (NOX2), the enzyme complex min before shearing the samples 10 times through a 28-1/2–gauge needle defective in CGD, is activated by microbes and their products, and to lyse phagocytes. Samples were diluted in saline, spread on YPG agar normally generates superoxide that is transformed into a variety of plates, and incubated 3–4 d at 37˚C. In some cases, MDM were grown in microbicidal reactive oxygen species (ROS) like hydrogen per- 35-mm petri dishes, treated with IFN-g for 2 d, and infected with pre- oxide and hypohalous acids (18). Previous studies showed that opsonized G. bethesdensis for 1 wk before harvesting with Cellstripper. Cells were then subjected to cytospin, differentially stained, and imaged G. bethesdensis was less effective at activating normal PMN via light microscope. NOX2 than E. coli (13). We measured normal monocyte superoxide production in response to G. bethesdensis and E. coli using Cytokine analysis luminol-enhanced chemiluminescence. A dose-dependent luminol- Freshly isolated monocytes (1 3 105/200 ml/well) or MDM (5 3 104/200 enhanced chemiluminescence response occurred when normal ml/well) were not infected or infected with G. bethesdensis or E. coli at monocytes were incubated with G. bethesdensis (Fig. 2A, 2B). an MOI of 1 or 0.25, respectively, in 96-well plates (precoated with 10% autologous serum, as described above) in the presence of 10% autologous This response was dampened in the absence of serum (data not serum for 24 h. Supernatants were collected and frozen until analysis. shown), suggesting that phagocytosis of G. bethesdensis or Thawed supernatants were diluted 1:3, and cytokines were measured using phagocyte interaction with serum components such as comple- The Journal of Immunology 3299 Downloaded from http://www.jimmunol.org/

FIGURE 1. Serum-dependent internalization of G. bethesdensis by CD14+ monocytes. (A) CD14+ monocytes from normal (n = 5–12) and CGD (n =5) donors were incubated with G. bethesdensis at an MOI of 10 bacteria per host cell for 1 h in the absence of serum or presence of autologous serum or heat- treated (HT, 56˚C, 30 min) serum, as described in Materials and Methods (mean 6 SD, nonparametric t tests where **p # 0.01). Internalization of bacteria by guest on September 26, 2021 by CGD monocytes in the absence of serum was not determined. (B) Confocal microscopy imaging of normal CD14+ monocytes incubated with pHrodo- labeled G. bethesdensis (red) at an MOI of 10 bacteria per host cell for 1 h in the presence of autologous serum or heat-treated serum. Nuclei are labeled with DAPI (blue). Bar length, 50 mm. (C) CD14+ monocytes from normal (n = 3) and CGD (n = 3) donors were incubated with pHrodo-labeled G. bethesdensis at an MOI of 1 bacterium per host cell for the indicated time points in the presence of autologous serum. Epifluorescence images were scored, and data were represented as mean 6 SD. ment is at least partially required for the generation of superoxide we hypothesized that monocytes would also be able to kill G. in monocytes. E. coli was equivalently stimulatory compared with bethesdensis. Normal monocytes killed G. bethesdensis in a dose- G. bethesdensis (Fig. 2B), but the kinetics of the response differed dependent manner after 24 h of infection (Fig. 3A). In contrast, (Fig. 2A). The relative lag in response to G. bethesdensis (Fig. 2A) CGD monocytes were incapable of killing G. bethesdensis and, mirrored the kinetics of internalization (Fig. 1C). moreover, provided a suitable environment for growth of the or- In comparison with PMN, monocytes generated a weaker re- ganism (Fig. 3A). To exclude outgrowth of extracellular bacteria, sponse to G. bethesdensis and E. coli (Fig. 2C, 2D). This differ- gentamicin protection assays were performed. Although qualita- ence in respiratory burst has been previously shown for PMN and tivelysimilarresultswereobtained1hpostinfection,withor monocytes stimulated with E. coli, PMA, or zymosan (19–22). It without gentamicin, almost complete killing occurred in both should also be noted that E. coli induced a biphasic response in normal and CGD monocytes by 24 h after gentamicin washout, monocytes and PMN (Fig. 2A, 2C). Based on experiments in indicating that this organism is sensitive to gentamicin internal- which the stimulus was mixed in with the cells versus gently ized by monocytes prior to washing (data not shown). A com- added, it was determined that the first peak was due in part to parison of PMN and monocytes isolated from the same normal mechanical stimulation during initial stimulus addition, whereas donors showed that both cell types killed similar amounts of G. the second peak occurred as a direct result of the stimulus inter- bethesdensis (Fig. 3B). Therefore, monocytes require the NADPH acting with the phagocytes (data not shown). Taken together, G. oxidase for the killing of G. bethesdensis and exhibit bactericidal bethesdensis induces a respiratory burst in normal monocytes and activity on par with PMN. to a greater extent in normal PMN. Treatment of monocytes with IFN-g significantly enhances their microbicidal activities (24–26), possibly through the upregulation NADPH oxidase-dependent killing of G. bethesdensis by + of NOX2 expression (27, 28). We tested whether treatment of CD14 monocytes normal monocytes with IFN-g for 2 d altered their ability to kill G. Stimulation of a respiratory burst in monocytes has been linked to bethesdensis. Whereas normal monocytes cultured for 2 d without killing of internalized organisms such as Staphylococcus aureus IFN-g lost their ability to kill, IFN-g treatment maintained the (23). Given that G. bethesdensis induces superoxide in monocytes, killing abilities of normal monocytes (Fig. 4A). Similar amounts 3300 G. BETHESDENSIS PERSISTENCE IN CGD MONOCYTES/MACROPHAGES Downloaded from http://www.jimmunol.org/

FIGURE 2. G. bethesdensis induces oxidative burst activity in normal CD14+ monocytes. (A) Luminol-enhanced chemiluminescence was measured in monocytes exposed to control buffer, G. bethesdensis,orE. coli at MOIs of 100 and 10 bacteria per host cell in the presence of autologous serum. Data are mean + SD for seven donors. (B) Data from (A) are represented as area under the curve (AUC). (C) Luminol-enhanced chemiluminescence was measured in PMN and monocytes [isolated from the same donors (n = 6)] exposed to control buffer, G. bethesdensis,orE. coli at an MOI of 100 bacteria per host cell in the presence of autologous serum. Data are mean + SD. (D) AUC data for PMN and monocytes, from the same donors (n = 6), exposed to bacteria at MOIs of 100 [shown in (C)] and 10 bacteria per host cell in the presence of autologous serum. by guest on September 26, 2021 of G. bethesdensis were recovered when the bacteria were incu- from CGD patients were responsive to IFN-g treatment, as indi- bated with PBS control or IFN-g in the absence of monocytes cated by significantly reduced bacterial burdens compared with (data not shown). G. bethesdensis-stimulated normal monocytes CGD monocytes that had not received the cytokine (Fig. 4A). pretreated with IFN-g produced significantly more superoxide, as Interestingly, the magnitude of the monocyte response to IFN-g measured by chemiluminescence, compared with monocytes not varied considerably from patient to patient. Previous studies in- receiving IFN-g (data not shown), suggesting a link between indicated that IFN-g treatment can enhance superoxide production duced superoxide and enhanced antibacterial activity after IFN-g in CGD monocytes if there are detectable levels of residual su- treatment. These data confirm that IFN-g treatment is important peroxide at baseline (29, 31). Moreover, enhanced monocyte su- for the maintenance of in vitro monocyte bactericidal activity and peroxide production accompanied bactericidal activity against S. may augment phagocytic killing in vivo (29, 30). aureus for a CGD patient receiving in vivo IFN-g treatment (29). Given these previous studies, we predicted a priori a positive re- IFN-g stimulates some CGD monocytes to control lationship between residual PMN ROS production (32) and mono- G. bethesdensis growth cyte killing of G. bethesdensis. Thus, we employed a one-tailed Because IFN-g maintains normal monocyte killing of G. bethes- p value to test this prediction. A Spearman correlation demon- densis, we next tested the capacity of IFN-g–treated CGD mono- strated a positive relationship (r = 0.442) between reduction in cytes to kill G. bethesdensis. When considered as a group, monocytes bacterial burden and CGD neutrophil residual superoxide that

FIGURE 3. Freshly isolated normal, but not CGD CD14+ monocytes kill G. bethesdensis. (A)CD14+ monocytes from normal (n =9)andCGD(n = 3) donors were incubated with G. bethesdensis at MOIs of 2, 1, and 0.5 bacteria per host cell in the presence of autologous serum for 24 h. Data are represented as mean + SD. Linear regression analysis was conducted to assess differences in slope of lines (*p # 0.05) for normal versus CGD. (B) PMN and monocytes isolated from the same normal donors (n = 8) were incubated with G. bethesdensis at an MOI of 1 bacteria per host cell in the presence of autologous serum for 24 h. Data are represented as percentage of control input (mean 6 SD). The Journal of Immunology 3301

infection (33). Culturing CD14+ monocytes in the presence of M-CSF for 1 wk results in a macrophage-like phenotype (34). Whereas normal MDM enabled growth of G. bethesdensis, treatment of these cells for 2 d with IFN-g generated MDM capable of controlling G. bethesdensis growth in a dose-dependent manner (Fig. 6A). CGD MDM were significantly less effective in controlling G. bethesdensis growth either in the absence or presence of IFN-g (Fig. 6B). We confirmed via confocal microscopy that normal and CGD MDM internalized similar amounts of bacteria at various MOIs in the presence of serum after 1 h (data not shown). Macrophages are highly phagocytic cells that support the FIGURE 4. IFN-g maintains microbicidal activity of normal monocytes persistence of several bacterial pathogens such as Salmonella and stimulates monocytes from some CGD subjects to control G. bethes- typhimurium, Mycobacterium tuberculosis, Legionella pneumophila, densis growth. (A) Monocytes from normal (n = 24) and CGD (n = 16) and Brucella species (35–37). Given that normal MDM were in- donors were treated with PBS control or 65 U IFN-g/ml for 2 d before capable of controlling G. bethesdensis growth at the MOIs tested infection with G. bethesdensis for 24 h at an MOI of 1 bacterium per host after 24 h, unless supplemented with IFN-g, we examined the cell in the presence of autologous serum. Data are represented as per- survival of these bacteria after 1 wk of coculture with MDM. IFN- centage of control input. Significance testing of PBS control versus IFN-g Downloaded from treatment was by Wilcoxon paired t test (****p # 0.0001). (B) Percentage g–treated normal MDM infected for 1 wk killed G. bethesdensis at decrease in bacterial burden after IFN-g treatment relative to PBS control an MOI of 0.25 bacteria per host cell (Fig. 7A, 7B). However, for all normal and CGD monocytes in (A). Percentages for CGD mono- IFN-g–treated CGD MDM maintained a favorable environment cytes are plotted against residual superoxide in PMN from the same for G. bethesdensis survival (Fig. 7B, 7C). No decrease in via- CGD patients. The relationship of the two variables was assessed using a bility, as assessed by lactate dehydrogenase assay and XTT assay, Spearman correlation, and the best fit (Y = 6.4093 + 21.59) is depicted. was observed for normal or CGD MDM infected with G. bethes-

densis for 1 wk compared with noninfected MDM incubated for http://www.jimmunol.org/ was statistically significant (one-tailed p value = 0.044) (Fig. 4B). the same time period (data not shown). Based on these findings, The genetic background of these CGD patients is described in the CGD macrophage may contribute to the persistence of this Table I. Understanding the mechanism of IFN-g–induced bac- bacterium. tericidal activity of CGD monocytes could prove a powerful diag- G. bethesdensis induces cytokine release from normal and nostic tool to determine the likelihood that IFN-g treatment will CGD monocytes and MDM benefit specific CGD patients. Proinflammatory cytokines initiate host responses during infection, G. bethesdensis infection does not affect monocyte survival whereas anti-inflammatory cytokines play a significant role during

We next assessed the impact of G. bethesdensis infection on the resolution phase once infection has been contained. Increased by guest on September 26, 2021 monocyte viability using an XTT assay, which measures the re- proinflammatory and decreased anti-inflammatory cytokine production of XTT to an orange formazan product by metabolically duction have been reported for activated CGD cells compared with active cells. No significant difference was detected between nor- activated normal cells in some settings (16, 38), whereas, in others, mal and CGD monocytes either uninfected or incubated for 24 h the opposite trend was observed (39). We measured cytokine con- with G. bethesdensis at MOIs of 0.5:1, 1:1, and 10:1 in the centrations in supernatants from normal and CGD monocytes presence of serum (Fig. 5), indicating that monocytes remain vi- and MDM cultured with G. bethesdensis or E. coli for 24 h. Both able in the presence of G. bethesdensis. bacteria induced proinflammatory and anti-inflammatory cytokine release from all cell types tested with E. coli eliciting significantly G. bethesdensis persistence in CGD MDM greater responses than G. bethesdensis (Table II). No significant Peripheral blood monocytes are precursors to macrophages, sup- differences could be detected between noninfected normal and plying tissues during steady-state conditions or in the setting of CGD cell cultures. However, CGD monocytes produced signifi-

Table I. CGD patient subtype and speciﬁc mutation

CGD Patient Gene Affected DNA Mutation Protein Mutation Mutation Type 1 gp91 c.676 C . T p.Arg2263 Nonsense 2 gp91 c.483 + 1 G . C Del exon 5 Splice 3 gp91 c.80-83 del TCTG p.Val27GlyfsX33 Frameshift 4 gp91 c.742 dup A p.Ile248fsX37 Frameshift 5 gp91 c.868 C . T p.Arg2903 Nonsense 6 gp91 c.676 C . T p.Arg2263 Nonsense 7 gp91 c.676 C . T p.Arg2263 Nonsense 8 gp91 c.675-1 G . A Del exons 6 & 7 Splice 9 p47 Not determined Not determined Not determined 10 gp91 c.909 C . A p.His303Gln Missense 11 gp91 c.388 C . T p.Arg1303 Nonsense 12 p47 c.75_76 del GT p.Tyr26fsX26 Frameshift 13 gp91 c.868 C . T p.Arg2903 Nonsense 14 p47 c.75_76 del GT p.Tyr26HisfsX26 Frameshift 15 p47 Not determined Not determined Not determined 16 p47 c.75_76 del GT p.Tyr26HisfsX26 Frameshift 3302 G. BETHESDENSIS PERSISTENCE IN CGD MONOCYTES/MACROPHAGES

tide (13). Serum resistance has been linked to increased virulence in other Gram-negative bacilli such as L. pneumophila strain UH1 (43), O-Ag–positive B. abortus strains (44), and many others. Virulence factors also enable L. pneumophila (45, 46), M. tuberculosis (47), and Brucella species (48) to resist killing by a variety of cationic antimicrobial peptides that compose a major ﬁrst line of host defenses. G. bethesdensis may also come into contact with innate immune phagocytes. One of the ﬁrst cellular responders recruited to the site of bacterial infection is the neutrophil. In vitro studies show that healthy neutrophils internalize G. bethesdensis in a comple- FIGURE 5. G. bethesdensis infection does not alter monocyte survival. ment-dependent manner and kill ∼50% of input bacteria after 24 h Monocytes from normal (n = 6) or CGD (n = 3) donors were incubated of infection at an MOI of 1 (13). CGD neutrophils internalize G. alone or with G. bethesdensis at MOIs of 0.5, 1, or 10 bacteria per host cell bethesdensis normally, but fail to kill the organism and instead in the presence of autologous serum for 24 h. Cell viability was assessed control its growth, highlighting the importance of the NADPH using an assay that measures conversion of XTT to an orange formazan oxidase in G. bethesdensis killing by PMN. Survival of this bac- product by metabolically active cells (mean OD + SD). terium in neutrophils may be further enhanced by the ability of G. bethesdensis to inhibit neutrophil apoptosis (13), which would cantly more cytokine than normal monocytes when infected with normally aid in the containment and clearance of the microbe by Downloaded from either bacterium, whereas MDM responses were similar in mag- macrophages. Although the neutrophil may play an important role nitude for normal versus CGD. There was little to no production of in survival of G. bethesdensis, it is relatively short-lived [∼5- to IL-1b (for MDM), IL-2, IL-4, IL-5, IL-7, IL-12p70, IL-13, and 8-h t1/2 in circulation (49)] compared with other innate immune GM-CSF (data not shown). Taken together, the lower cytokine phagocytes like monocytes [∼3-d t1/2 in circulation (50)] and responses of monocytes and MDM to G. bethesdensis, compared macrophages [∼30-d t1/2 (51)].

with E. coli, may help G. bethesdensis persist in these cells. Monocytes are recruited to the site of infection and undergo http://www.jimmunol.org/ differentiation to macrophages within the infected tissue, which Discussion also houses resident macrophages. We demonstrate in this study G. bethesdensis is a bacterial pathogen that can infect patients that monocytes from healthy and CGD donors internalize G. with CGD. Isolation of genetically indistinguishable strains from bethesdensis. Although normal PMN internalization of G. bethes- individual subjects over 2–3 y, despite periods without clinically densis relies almost entirely on a temperature-sensitive serum evident infections, may reﬂect either reinfection or reactivation of component (13), normal monocyte internalization only partially a latent infection. Persistence of bacteria has been suggested for depends on a heat-labile serum component. This difference in a number of human diseases, including Legionnaires’ disease (L. phagocytic mechanism may be explained by the differential ex- pneumophila) (40), tuberculosis (M. tuberculosis) (41), and bru- pression of FcRs on the two cell types (52) or the wider repertoire by guest on September 26, 2021 cellosis (Brucella species) (42). G. bethesdensis shares several of receptors [e.g., scavenger receptors (53)] on monocytes. Dif- attributes with these microbes that may explain its ability to sur- ferential expression or activation of phagocytic receptors, as well vive in the host for a long period of time, as follows: 1) resistance as pattern recognition receptors (PRRs) (54, 55), on PMN and to killing by nonoxidative host defenses; 2) replication and sur- monocytes may also explain the difference in relative magnitude vival inside host cells; and 3) promotion of host cell survival. of the respiratory burst in these two cell types, and in response to The natural reservoir for G. bethesdensis and the mode of entry E. coli versus G. bethesdensis. For instance, PRRs dectin-1 and into the human host are unknown. Regardless of the site of entry, dectin-2 on inﬂammatory PMN and monocytes/macrophages have G. bethesdensis must resist a variety of innate immune host de- been shown to differentially contribute to fungal particle associ- fenses to establish infection. G. bethesdensis is resistant to killing ation and subsequent ROS production (56). The same study also by human serum and the cathelicidin cationic antimicrobial pep- showed that serum opsonization of zymosan or Candida albicans

FIGURE 6. Normal but not CGD MDM control G. bethesdensis growth after 24 h of infection. (A) Normal MDM were incubated 6 65 U IFN-g/ml for 2 d before coculture with G. bethesdensis for 24 h at the indicated MOI in the presence of autologous serum. Data are presented as mean + SD (n =11 donors). Linear regression analysis was conducted to assess differences in slope of lines (**p # 0.01) for PBS control versus IFN-g treatment. (B) MDM were treated as in (A) at an MOI of 0.25 bacteria per host cell. Data are percentage of control input for 11 normal donors and 5 CGD donors. Wilcoxon paired t test was used to compare PBS control versus IFN-g treatment (***p # 0.001). The Journal of Immunology 3303 Downloaded from http://www.jimmunol.org/

FIGURE 7. G. bethesdensis persists inside CGD MDM for 1 wk. (A) Normal MDM were incubated 6 65 U IFN-g/ml for 2 d before coculture with G. bethesdensis for 1 wk at the indicated MOI in the presence of autologous serum. Data are presented as mean + SD (n = 6 donors). Linear regression analysis was conducted to assess differences in slope of lines (*p # 0.05) for PBS control versus IFN-g treatment. (B) MDM were treated as in (A) at an MOI of 0.25 by guest on September 26, 2021 bacteria per host cell. Data are percentage of control input for six normal donors and six CGD donors. Wilcoxon paired t test was used to compare PBS control versus IFN-g treatment (*p # 0.05). (C) IFN-g–treated normal and CGD MDM infected for 1 wk with G. bethesdensis at an MOI of 1 bacterium per host cell. Black arrows indicate the presence of bacteria. was important for interaction with PMN, but less so for mono- G. bethesdensis even with the help of IFN-g. Our studies dem- cytes/macrophages. onstrate that four normal IFN-g–treated MDM are required to One downstream effect of phagocytosis and PRR activation is the control the growth of a single bacterium after 24 h of infection, production of cytokines, which contribute to the inflammation and and only modest killing occurs by 1 wk postinfection. This in- resolution phases of infection. In this study, we show that G. ability to kill G. bethesdensis is more pronounced in CGD MDM bethesdensis induces cytokine release from normal and CGD in which IFN-g could not significantly control intracellular rep- monocytes and macrophages, but to a significantly lesser extent lication of the organism up to 1 wk postinfection. than E. coli. Suppressing cytokine responses can be a microbial These data suggest that the CGD macrophage is a potential niche evasion mechanism as in the case of L. pneumophila,which for G. bethesdensis growth and survival in vivo. The macrophage requires a type II secretion system to dampen cytokine release as a niche for bacterial replication and persistence is not unprec- from and permit growth within infected monocytes (57), although edented. Alveolar macrophages internalize L. pneumophila via such mechanisms have yet to be described for Granulibacter. Our conventional complement-mediated phagocytosis (58) or coiling laboratory is currently characterizing structural differences be- phagocytosis (59) into phagosomes that permit bacterial replica- tween G. bethesdensis and E. coli endotoxin that may also con- tion and evade lysosomal fusion (60). This suitable environment tribute to the observed differences in cytokine secretion. for L. pneumophila replication may allow the organism to survive Similar to neutrophils from normal subjects, freshly isolated and for a long period of time. Moreover, L. pneumophila inhibits IFN-g–treated normal monocytes kill similar amounts of input macrophage apoptosis by blocking the effects of proapoptotic bacteria after 24 h of infection at an MOI of 1. However, unlike Bcl2 family members (61). Latency of the organism in patients CGD neutrophils (13), freshly isolated CGD monocytes allow a 2- from the 1976 Philadelphia epidemic of Legionnaires’ disease has to 3-fold expansion of G. bethesdensis over 24 h, demonstrating been suggested, in which specific IgM Abs persisted for 2 y after that these cells provide a favorable environment for survival of the initial infection (40). bacterium. Although it does not induce killing, IFN-g treatment of Latent infection by M. tuberculosis in one-third of the human CGD monocytes significantly limits G. bethesdensis outgrowth, population has been reported (41). Infected macrophages play and this effect correlates with the amount of residual ROS in a central role in latency by forming granulomas with Ag-specific CGD neutrophils. In comparison with neutrophils and monocytes, T cells that inhibit the growth of M. tuberculosis, whereas the MDM are less successful in exerting bactericidal activity against bacterium resists macrophage bactericidal killing (62). Like L. 3304

Table II. Cytokine proﬁles of normal and CGD monocytes and MDM infected with G. bethesdensis or E. coli for 24 h

Normal (n = 7) CGD (n =4) p Values

Cytokinea (I) Control (II) G. bethesdensis (III) E. coli (IV) Control (V) G. bethesdensis (VI) E. coli II versus IIIb V versus VIb II versus Vc III versus VIc Monocytes IL-1b 103 (52) 608 (126) 5,129 (2,502) 74 (84) 1,088 (462) 14,244 (3,620) * 0.13 ** * IL-6 278 (180) 5,404 (2,619) 13,462 (4,781) 2,605 (3,102) 26,699 (8,989) 31,512 (18,983) * NS ** * IL-8 15,089 (7,437) 22,252 (3,276) 27,901 (6,308) 9,794 (8,272) 20,904 (1,738) 27,254 (5,620) 0.08 0.13 NS NS IL-10 1 (1) 38 (47) 127 (94) 9 (13) 539 (147)+ 528 (469) * NS ** ** IL-17 29 (14) 76 (13) 111 (12) 37 (33) 93 (11) 134 (13) * 0.13 0.11 * G-CSF 10 (4) 217 (160) 662 (360) 73 (108) 2,310 (895) 1,938 (1,495) * NS ** * BETHESDENSIS G. IFN-g 82 (45) 437 (119) 815 (208) 177 (187) 714 (145) 1,154 (130) * 0.13 * ** CCL-2 1,587 (1,390) 3,004 (950) 1,435 (1,047) 2,334 (2,218) 2,990 (794) 873 (569) * 0.13 NS NS CCL-4 1,016 (601) 3,974 (1,582) 7,047 (2,495) 2,275 (2,216) 5,155 (485) 7,634 (3,947) * NS NS NS TNF-a 101 (87) 2,613 (2,178) 11,868 (5,249) 223 (241) 6,814 (6,075) 39,407 (12,606) * 0.13 * **

Normal (n = 5–7) CGD (n = 4–5) p Values

(I) Control (II) G. bethesdensis (III) E. coli (IV) Control (V) G. bethesdensis (VI) E. coli Cytokined (n =7) (n =7) (n =5) (n =5) (n =5) (n = 4) II versus IIIb V versus VIb II versus Vc III versus VIc MONOCYTES/MACROPHAGES CGD IN PERSISTENCE MDM IL-6 2 (2) 84 (95) 1,890 (1,904) 4 (2) 60 (78) 2,834 (1,912) ** * NS NS IL-8 185 (117) 1,885 (1,604) 6,667 (4,561) 436 (418) 3,264 (3,083) 11,548 (6,601) * 0.06 NS NS IL-10 2 (2) 10 (11) 214 (182) 5 (3) 13 (10) 940 (747) * * NS 0.11 G-CSF 2 (1) 6 (4) 81 (48) 3 (1) 8 (4) 184 (137) ** * NS NS IFN-g 4 (5) 35 (32) 151 (85) 20 (15) 47 (35) 198 (91) * * NS NS CCL-2 547 (398) 972 (880) 1,318 (862) 1,979 (1,104) 2,865 (1,931) 2,602 (1,862) NS NS * NS CCL-4 139 (210) 902 (1,077) 6,330 (10,096) 136 (71) 1,111 (1,476) 8,350 (6,852) NS 0.11 NS NS TNF-a 3 (2) 118 (114) 5,502 (6,627) 5 (4) 115 (132) 8,274 (6,333) ** * NS NS aCytokines from 5 3 105 monocytes/ml infected at an MOI of 1 are represented as mean (SD) (pg/ml). bStatistical comparisons by exact paired nonparametric Wilcoxon signed rank test, *p # 0.05. cStatistical comparisons by exact unpaired nonparametric Mann–Whitney U test, *p # 0.05, **p # 0.01.

dCytokines from 2.5 3 105 MDM/ml infected at an MOI of 0.25 are represented as mean (SD) (pg/ml).

Downloaded from from Downloaded http://www.jimmunol.org/ by guest on September 26, 2021 26, September on guest by The Journal of Immunology 3305 pneumophila, M. tuberculosis also inhibits phagosomal maturation (79); however, we found no contribution of vitamin D in the and replicates within macrophages (63). Additionally, its virulence control of G. bethesdensis burden in vitro (data not shown). It will factors are critical for the inhibition of macrophage apoptosis (64) be important to establish further correlations between in vitro and and pyroptosis (65). Lastly, O-Ag–positive Brucella species also in vivo IFN-g efficacy to help identify specific CGD patients who prevent phagosome–lysosome fusion inside macrophages (66) and will respond to treatment. Finding novel therapies to treat chronic inhibit macrophage apoptosis (67), which most likely prolongs intracellular infections, especially in immunocompromised pop- the bacterium’s intracellular survival. Cases of chronic brucello- ulations, is crucial given that many persistent bacterial pathogens sis have been described as lasting from several months to several are resistant to antibiotics. years after acute infection (42, 68). Taken together, the macrophage centrally contributes to the persistence of bacteria that cause Acknowledgments chronic disease in humans. We acknowledge Drs. Harry L. Malech, Suk See DeRavin, and Stephen M. G. bethesdensis causes recurrent infections in CGD patients for Holland for generously providing us with CGD patient samples, and we years and can be recovered from normal mouse spleens up to thank the CGD patients who have contributed to this study. We also thank 76 d postinoculation (3, 4). Like the other persistent bacterial Drs. Margery Smelkinson and Stephen Becker for assistance with epifluo- pathogens mentioned above, G. bethesdensis resists killing by rescence and confocal microscopy, Anthony Pettinato for blindly scoring serum and cationic antibacterial peptides, replicates within CGD monocyte internalization images, Drs. Mark VanRaden and Chiung-Yu macrophages, and does not compromise the viability of infected Huang for statistical help, Neysha Martinez for preliminary studies provok- macrophages. G. bethesdensis resists some oxygen-independent ing us to pursue this study, and Dr. David Greenberg for helpful suggestions microbicides, but it may not be impervious to all nonoxidative during the beginning of this study and critical review of this manuscript. Downloaded from killing mechanisms. Although the NADPH oxidase is required for G. bethesdensis clearance, it is possible that the products of the Disclosures NADPH oxidase interact with and activate oxygen-independent The authors have no financial conflicts of interest. bactericidal systems. Interestingly, all of the aforementioned bacteria have slow rates References of division with G. bethesdensis doubling every 5–6 h, L. pneu- http://www.jimmunol.org/ 1. Winkelstein, J. A., M. C. Marino, R. B. Johnston, Jr., J. Boyle, J. Curnutte, mophila every 3 h (69), M. tuberculosis every 24 h (70), and J. I. Gallin, H. L. Malech, S. M. Holland, H. Ochs, P. Quie, et al. 2000. Chronic Brucella species every 2.5–3.5 h (71), which contrasts to E. coli’s granulomatous disease: report on a national registry of 368 patients. Medicine faster doubling time of 0.5–1.5 h (72). This trait could be im- 79: 155–169. 2. Song, E., G. B. Jaishankar, H. Saleh, W. Jithpratuck, R. Sahni, and G. 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