Apoptosis are induced in J774 macrophages upon phagocytosis and killing of Pseudomonas aeruginosa

Cellular Immunology 286 (2013) 11–15

Contents lists available at ScienceDirect

Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm

Apoptosis are induced in J774 macrophages upon phagocytosis and killing of Pseudomonas aeruginosa Jianling Zhang a,b,⇑, Ru Jiang c, Wei Wang d, Hisao Takayama b, Yoshinori Tanaka b a

Department of Immunology and Pathogenic Biology, Tianjin University of Traditional Chinese Medicine, 312 West Anshan Road, Nankai District, Tianjin 300193, China Department of Microbiology and Pathology, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan c Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA d Department of Biochemistry, Tianjin University of Traditional Chinese Medicine, 312 West Anshan Road, Nankai District, Tianjin 300193, China b

a r t i c l e

i n f o

Article history: Received 24 June 2013 Accepted 25 October 2013 Available online 6 November 2013 Keywords: Apoptosis Caspase 3 Macrophage Phagocytosis Pseudomonas aeruginosa

a b s t r a c t Apoptosis induced by Pseudomonas aeruginosa in host cells plays a role in pathogenesis. However, little is known how the apoptosis of macrophages harboring P. aeruginosa affects the host or pathogen. In this study, the viability of J774 macrophages phagocytosing Pa IID1117 (elastase- and protease-positive) was significantly reduced (53.8 ± 4.5%) 48 h after infection and cell death occurred via apoptosis as seen by Hoechst 33258 staining and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) assay. An activated subunit of caspase 3 was found in the cellular lysate. Lower colony counts in infected cells and effective intracellular killing of bacteria were accompanied by enhanced apoptosis. Caspase 3 inhibiter inhibited apoptosis but did not prevent cell death and the extracellular leakage of bacteria. The apoptosis of the macrophages that phagocytose P. aeruginosa therefore inhibits the intracellular growth and spread of this bacterium and is important in host defense against P. aeruginosa infections. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes nose, throat, and respiratory tract infections, and soft tissue infections, particularly in patients with cystic fibrosis, cancer, AIDS, and severe burns [1–3]. The mechanism responsible for P. aeruginosa infection is related to the ability of the pathogen to interfere with the host defense mechanism components including host macrophages [4], neutrophils [5], and T lymphocytes [6]. Macrophages play a role in clearing P. aeruginosa from the host, including phagocytosis, antigen presentation, and cytokine induction [7–9]. However, P. aeruginosa also produces many virulence factors that damage host macrophages, thereby allowing the pathogen to colonize, penetrate, and survive [1,4–6,10]. In this type of pathogenesis, the induction of apoptosis by P. aeruginosa plays a key role. P. aeruginosa induces ExoU-independent type-III-secre-

tion-mediated apoptosis in macrophages [11]. In addition, bacterial azurin and N-3-oxododecanoyl homoserine lactone also accelerate apoptosis in macrophages [12]. Macrophage death caused by cell-surface-associated [5] and extracellular virulence factors [11,12] contributes to pathogen persistence and dissemination within the body. However, little is known about the apoptosis of macrophages that phagocytose P. aeruginosa and whether it benefits the host or pathogen. In the study, we infected J774 macrophages with Pa IID1117, a protease- and elastase-positive P. aeruginosa strain, and Pa IID1130, a protease-negative and elastase-positive strain. After phagocytosing the Pa IID1117 strain, the macrophages underwent apoptosis, which subsequently restricted bacterial survival. 2. Materials and methods 2.1. Macrophage cell line

Abbreviations: DMEM, Dulbecco’s Modified Eagle’s medium; FCS, fetal calf serum; Pa, P. aeruginosa; PBS, phosphate-buffered saline; MOI, multiplicity of infection; TUNEL, terminal deoxynucleotidyl transferase-mediated d-UTP-biotin nick end labeling; LDH, lactate dehydrogenase; SDS–PAGE, sodium dodecyl sulfate– polymerase chain reaction; CFU, colony-forming unit. ⇑ Corresponding author at: Department of Immunology and Pathogenic Biology, Tianjin University of Traditional Chinese Medicine, 312 West Anshan Road, Nankai District, Tianjin 300193, China. E-mail address: [email protected] (J. Zhang). 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.10.006

Macrophage cell line J774 was kindly supplied by Dr. Barry. R. Bloom of Harvard School of Public Health. The macrophages were cultured in Dulbecco’ Modified Eagle’s Medium (DMEM) (Nissui Pharmaceutical Co, Japan) supplemented with 10% fetal calf serum (FCS) and 50 U of penicillin–streptomycin per ml at 37 °C in the presence of 5% CO2 overnight. In some experiments, the cells were cultured in culture dishes containing glass slides. Following three

12

J. Zhang et al. / Cellular Immunology 286 (2013) 11–15

washes with phosphate-buffered saline (PBS), the cells were cultured in fresh medium before use in the various assays [13]. 2.2. Pseudomonas aeruginosa P. aeruginosa strain IID1117 (Pa IID1117) and IID1130 (Pa IID1130) were kindly provided by the Institute of Medical Science, University of Tokyo. Pa IID1117 is elastase-positive and proteasepositive and forms mucoid colonies, and Pa IID1130 is elstase-positive and protease-negative and also forms mucoid colonies [14]. The strains were grown in 8 ml of Tryptic Soy Broth (Becton Dickinson and Company Sparks, CA, USA) at 37 °C overnight with shaking. The harvested bacteria were washed three times with PBS and used in experiments. Some harvested bacteria were killed by heat (1 h, 56 °C) and resuspended in 8 ml of PBS before use.

2.6. Detection of caspase activation by Western blot analysis Cells were harvested, and washed with ice-cold PBS, and lysed on ice for 30 min in tritonX-100 lysis buffer (1% tritonX-100, 50 mM Tris–HCl [PH 7.4], 150 mM NaCl, 2 mM EDTA, 10% sucrose, 1 mM PMSF) containing Complete-mini protease inhibitor cocktail tablets (Roche Applied Science, Germany). Caspase 3 was then determined as previously described [13]. Briefly, the cell extract proteins were separated by 15% SDS–PAGE and electrotransferred to a nitrocellulose membrane (Millipore, Bedford, MA, U.S.A.). The membrane was incubated with anti-caspase-3 antibody (Cell Signaling Technology, MA, USA.) and alkaline phosphatase-conjugated secondary antibody (Boehringer Mannheim, Germany), the chemiluminescence was initiated and the membrane was exposed to an X-ray film [13].

2.3. Uptake and intracellular killing assays

2.7. Electron microscopy

J774 macrophages (1  104 cell/well) were cultured in 96-well tissue culture plates overnight, resuspended in medium containing 5% FCS, and infected with the Pa IID1117 and Pa IID1130 strains at multiplicity of infection (MOI) of 25 in DMEM at 37 °C in the presence of 5% CO2 for 1 h. In some experiments, the macrophages were pretreated with cytochalasin D (Sigma) for 30 min or with 50 lM caspase 3 inhibitor (Z-DEVD-FMK) (Roche) for 60 min, and then infected. After washing, gentamicin (200 lg/ml) was added and the macrophages were further incubated for 2 h to kill extracellular bacteria [14]. The cells were then lysed with 100 ll 0.25% Triton X-100 (Wako, Japan) to determine the number of viable intracellular bacteria at different times as previously described [14]. In some experiments, after the wells were washed three times, an equal number of uninfected macrophages (1  104 cell/ well) were added to infected macrophage monolayers 12 h after infection, and intracellular bacteria were determined as described above [14]. The cell viability was assessed at different times by trypan blue dye exclusion method. Three hundred cells on each slide were counted and every experiment was performed in triplicate [13].

J774 macrophages were infected with P. aeruginosa, and the cells were fixed for 24 h in 2% glutaraldehyde in PBS (PH 7.4). The cells were then washed in 7.5% sucrose-PBS buffer and dehydrated by a graded ethanol series. They were then embedded in Spurrs resin, thin sectioned, stained with 0.25% uranyl acetate and lead citrate, and examined with a model H-7100 transmission electron microscope (Hitachi, Japan). Photographs were taken on Kodak electron imaging film.

2.4. Morphological observation of nuclear change and TUNEL assay Macrophages that adhered to the coverslips were washed with PBS at different times and fixed with paraformaldehyde (4.5%) (Wako, Japan). The cells were then stained with Hoechst 33258 (Sigma), as previously described [13], and the nuclei were observed under a fluorescence microscope. Other slides were permeabilized with 0.5% TritonX-100 for 2 min at room temperature. The samples were then labeled using an in situ cell death detection kit, MEBSTAIN Apoptosis Kit II (Medical and Biological Laboratories, Nagoya, Japan) according to the manufacture’s protocol, and observed under a fluorescence microscope [13].

2.8. Statistical analysis Results were expressed as means ± standard deviation. Statistical differences were determined by Student’s t test, and a P value below 0.05 was considered to be statistically significant.

3. Results 3.1. Viability of J774 macrophages infected with P. aeruginosa To examine the effect of phagocytosed P. aeruginosa on macrophages, macrophage monolayers were infected with Pa IID 1117 and Pa IID 1130 strains at an MOI of 25. After 1 h, gentamicin was added to the medium to kill extracellular bacteria. Cell viability was monitored at different times by trypan blue exclusion assay. As shown in Fig. 1, cell viability was 53.8 ± 4.5% for Pa IID1117, 96.4 ± 1.7% for heat-killed Pa IID1117, 97.1 ± 2.3% for Pa IID1130, and 97.0 ± 1.2% for uninfected control cells 48 h after infection, respectively. The viability of cells harboring Pa IID1117 was significantly decreased (P < 0.01) compared with the control cells. After the cells were pretreated with cytochalasin D, a cellular actin microfilament inhibitor, cell death induced by Pa IID1117 was clearly inhibited (Fig. 1).

2.5. Lactate dehydrogenase (LDH) assay 3.2. Apoptosis of J774 macrophages phagocytosing Pa IID1117 Cells were infected with Pa IID1117 in the presence or absence of Z-DEVD-FMK as described above. The concentration of LDH released from the macrophages was measured using an LDH-cytotoxic test kit (Wako, Japan) according to the manufacture’s protocol. Briefly, cell-free supernatants were obtained at different times, an aliquot of the medium (50 ll) was added to the kit reagent and incubated for 45 min, and the reaction was then stopped and the absorbance measured at 560 nm using an ELISA plate reader. Released LDH was expressed as a percentage of total LDH activity. Independent experiments were performed in triplicate.

To determine whether the cell death described above was due to apoptosis, cells that phagocytosed the Pa IID1117 strain were evaluated for apoptotic markers. The cells were stained with Hoechst 33258 (Fig. 2A–C) and assessed by TUNEL assay (Fig. 2D–F). As shown in Fig. 2, cells infected with the Pa IID1117 strain exhibited chromatin condensation, apoptotic bodies (Fig. 2B), and fragmented DNA (Fig. 2E). In contrast, cells infected with Pa IID 1130 and uninfected cells had well-defined, almost spherical nuclei (Fig. 2A and C), and very few cells took up the label (Fig. 2D and F).

13

48

8h Pa I

ID 113

0,

.4 ID 111 7

Pa I

80

Pa I

Co ntr ol

Cleaved caspase 3 (17kDa) β-actin

48

Time (hour) Fig. 1. Cell viability of macrophages phagocytosed P. aeruginosa. J774 macrophages were incubated with Pa IID1117 in the presence (D) or absence (d) of cytochalasin D, heat-killed Pa IID1117 (h), Pa IID1130 () or in medium without bacteria (s). After the addition of gentamicin for 2 h, cell viability was assessed at different times by trypan blue dye exclusion method. The results are expressed as means ± standard deviation of three independent experiments. ⁄P < 0.01 versus control (heatkilled Pa IID1117 or Pa IID1130).

3.3. Caspase 3 is activated during apoptosis To investigate the intracellular mechanism responsible for apoptosis and whether cell death was induced through a caspase pathway, cell lysates were analyzed for the presence of the cleaved subunit of caspase 3 by Western blot at four different time points (2 h, 12 h, 24 h, and 48 h). As shown in Fig. 3, compared with the control and the cells infected with Pa IID1130, cleaved subunits (17 kDa) were released from procaspase 3 in the cells infected with Pa IID1117 (Fig. 3B) 12 h after infection and the relative expression of the cleaved subunits reached a maximum at 48 h (Fig. 3A and B). Because the active enzyme is composed of two subunits of 17 kDa and 12 kDa, caspase 3 was shown to be involved in the apoptosis induced by Pa IID 1117 strain. 3.4. Fate of P. aeruginosa in J774 macrophages To investigate the fate of intracellular Pa IID 1117 during apoptosis, P. aeruginosa were phagocytosed by macrophages and were

B

8h

,2

,4

72

ID 111 7

36

Pa I

24

ID 111 7

12

Pa I

0

ID 111 7

60

40

Pa I

*

4h

*

2h

*

60

,1

Viability (%)

A

ID 111 7

,2

h

100

h

J. Zhang et al. / Cellular Immunology 286 (2013) 11–15

Cleaved caspase 3 (17kDa) β-actin Fig. 3. Analysis of caspase 3 activation by Western blot. (A) J774 macrophages were incubated with Pa IID1117 for 2 h (lane 2) and 48 h (lane 3), Pa IID1130 for 48 h (lane 4), or in the medium without any bacteria (lane 1) for 48 h. (B) J774 macrophages were incubated with Pa IID1117 for 12 h (lane 1), 24 h (lane 2) and 48 h (lane 4). The cell lysates (50 lg of protein per sample) were subjected to Western blot analysis (15% SDS–PAGE gel) with anti-caspase 3 antibodies. (A) lane 3, (B) lane 1, 2 and 3 showed that activated subunit (17 kDa) was released. b-actin was included as a loading control.

observed to exist in the cells using a transmission electron microscope (Fig. 4). Intact P. aeruginosa within the phagosomes were observed 2 h after infection (Fig. 4B). After 48 h culture, the outer layer of P. aeruginosa lost its smooth surface and cellular integrity. P. aeruginosa were partially digested and accompanied by the nuclear condensation and fragmentation in the macrophages (Fig. 4C). The infected cells were lysed, and intracellular bacteria were enumerated by a colony counting method at different times. As shown in Fig. 5, the number of intracellular bacteria was reduced with increasing apoptosis. The number of the intracellular bacteria (Pa IID1117) was reduced 2 h after infection and active intracellular bacteria disappeared after 72 h (Fig. 5). The number of apoptotic cells was elected from 12 h of infection and reached 51.3 ± 5.5% at 72 h in the meantime (Fig. 5). The results indicated that the macrophages underwent apoptosis during the process of killing P. aeruginosa.

Fig. 2. Analysis of nuclear changes and DNA fragmentation in macrophages phagocytosed P. aerudinosa by Hoechst 33258 (A, B, C) staining and TUNEL assay (D, E, F). J774 macrophages were incubated with Pa IID1117 (B, E), Pa IID1130 (C, F), or in the medium without any bacteria (A, D) for 48 h. Apoptotic cells exhibited chromatin condensation and apoptotic bodies (B), and DNA fragmentation seen in yellow (E). Original magnification 400 for Hoechst 33258 staining; 200 for TUNEL assay.

14

J. Zhang et al. / Cellular Immunology 286 (2013) 11–15

A

B

C

60

60

4

50

50

40

3

30 2

20

1

10

0

0 0

12

24

36

48

60

72

Time (hour) Fig. 5. Number of intracellular bacteria and apoptotic cells. J774 macrophages (1  104 cells/well) were infected with Pa IID1117 (2.5  105 CFU/well) in DMEM containing 5% FCS. The cells were lysed and the number of intracellular bacteria was assessed by the colony counting method at different times (d). Fresh uninfected cells were added 12 h after infection (N). The number of apoptotic cells was assessed at different times by TUNEL assay (h).

LDH release (%)

5

Apoptotic cells (%)

CFU (log)

Fig. 4. Transmission electron microscopy of P. aeruginosa-infected macrophages. J774 macrophages were infected with Pa IID1117. (A) Control macrophages. (B) P. aeruginosa (arrows) were phagocytosed by macrophages 2 h after infection. (C) Partially digested P. aeruginosa 48 h after infection (the outer layer of P. aeruginosa lost its smooth surface and cellular integrity, arrow) and nuclear condensation and fragmentation were observed in the macrophages 48 h after infection. Bars, 1 lm.

∗ ∗

40

∗ ∗

30 20 10 0 0

12

24

36

48

60

72

Time (hour) Fig. 6. Percent LDH release from P. aeruginosa-infected macrophages. J774 macrophages were infected with Pa IID1117 in the presence (N) or absence (j) of caspase 3 inhibitor (Z-DEVD-FMK). Control cells were cultured in medium without any bacteria (s), and the culture supernatants were harvested at various times to determine LDH concentration. The results are expressed as means ± standard deviation of three independent experiments. ⁄P < 0.01 versus control or absence of Z-DEVD-FMK.

3.5. Macrophage apoptosis inhibits the spread of P. aeruginosa Fresh uninfected macrophages were added to the medium of infected cells 12 h after infection, and the fresh cells were observed to gather and adhere to P. aeruginosa-infected cells 6 h after addition (data not shown). Intracellular bacteria were eliminated after 48 h (Fig. 5). Furthermore, after the cells were pretreated with ZDEVD-FMK, an inhibitor of caspase 3, apoptosis was blocked but cells phagocytosing Pa IID1117 still died. Cell death by necrosis was also observed by LDH release assay (Fig. 6). Apoptotic macrophages not treated with Z-DEVD-FMK did not release more LDH into the medium compared with uninfected cells. Necrotic death led to intracellular bacteria release into the medium from 18 h, and bacteria was released into the medium of most all wells 24 h after infection. In contrast, the apoptotic macrophages not treated with Z-DEVD-FMK had limited leakage, and intracellular bacteria were not found to release into the medium for up to 72 h using the colony counting method.

4. Discussion In this study, we found that J774 macrophages which phagocytosed P. aeruginosa (Pa IID1117) underwent cell death. By Hoechst staining and TUNEL assay, the cell death was found to exhibit apoptotic characteristics including cell shrinkage, nuclear condensation, and the formation of apoptotic bodies [15].

Macrophages are a critical component of the innate immune response in humans, and eliminate invading microorganisms by phagocytosis. However, phagocytosed pathogens such as Shigella flexneri [16], Salmonella typhimurium [17], Yersinia enterocolitica [18], Escherichia coli [19], Mycobacterium avium [13,20], and Staphylococcus aureus [21] induce apoptosis by different mechanisms that involve the activation of caspases [13,22], up-regulation of endogenous receptor/ligand systems [23], mitochondrial signaling [24]. In our study, apoptosis induced by Pa IID1117 was associated with phagocytosis and intracellular killing of P. aeruginosa. Active P. aeruginosa was responsible for this apoptosis because heat-killed P. aeruginosa did not cause cell death and did not exhibit apoptosisinducing capability. In the presence of cytochalasin D, phagocytosis and cell death were inhibited. These results suggest that phagocytosed P. aeruginosa release some component(s) that initiates cell death or elevates expression of death receptors (Fas and TNFR) [21,22,24]. Pa IID1117 is an elastase- and protease-positive strain and PaIID1130 is an elastase-positive and protease-negative strain, and the two strains exhibit different releasing characteristics. Because the cells were not induced apoptosis after the elastase-positive Pa IID 1130 strain was phagocytosed, the apoptosis is suggested not to be related to elastase. Caspase, a family of cysteine proteases, play a critical role in the execution phase of apoptosis and are responsible for many of the biochemical and morphological changes associated with apoptosis,

J. Zhang et al. / Cellular Immunology 286 (2013) 11–15

and the caspase pathway is the most important of apoptotic pathway [25]. Fourteen caspases have so far been identified, some of which are directly involved in cellular apoptosis, which are known as effectors, especially caspase 3. From our results (Fig. 3), caspase 3 was activated in this process and apoptosis was inhibited by ZDEVD-FMK, a cell-permeable, potent inhibitor of caspase 3, apoptosis induced by P. aeruginosa therefore depends on a caspase signaling pathway. Because caspase 3 is a central effector caspase in the apoptotic pathway and in our study, we observed that caspase 3 was blocked by Z-DEVD-FMK in the infected cells with P. aeruginosa, apoptosis in infected macrophage was inhibited and cellular mortality significantly decreased. The modulation of apoptosis plays a role in the pathogenesis of a diverse group of infectious diseases including bacterial, viral, and protozoan infections [26]. Macrophage apoptosis induced by pathogenic bacteria is a bacterial strategy used to avoid immune-mediated killing. Macrophage apoptosis may be a pathogen-directed mechanism used for immune avoidance by pathogens, including P. aeruginosa [27]. However, we found that macrophages which phagocytosed P. aeruginosa underwent apoptosis, which the growth of intracellular bacteria was inhibited, and that lower colony counts and effective intracellular bacteria killing was accompanied by enhanced apoptosis. Apoptotic cells limited intracellular P. aeruginosa escape into the extracellular medium, since there were almost no bacteria present in extracellular medium in the infection experiment. After the addition of the caspase 3 inhibiter, apoptosis was inhibited but macrophages that phagocytosed P. aeruginosa died due to necrosis. Intracellular bacteria were released into the extracellular medium in almost all wells 18 h after infection. The results showed that apoptosis but not necrosis is associated with the killing, which limited the growth and spread of intracellular P. aeruginosa. In previous reports on P. aeruginosa-induced macrophage apoptosis, macrophage apoptosis was regarded to benefit pathogen colonization, penetration, and survival in the host. We believe that apoptosis induced by P. aeruginosa should be divided into three classes: First, apoptosis induced by extracellular virulence factors released by Type I and II secretion system [11,12,14]; Second, apoptosis induced by cell-to-cell contact by Type III secretion system [5]; Third, apoptosis induced by phagocytosed P. aeruginosa. For the first two classes, pathogen virulence factors were released into extracellular medium or cytoplasm to cause macrophage death that enables the pathogen to evade death by immune cells and to persist and disseminate in the host. However, apoptosis of macrophages phagocytosing P. aeruginosa might be a very useful mechanism and important in host defense against P. aeruginosa infections. Acknowledgments We thank Dr. Barry R. Bloom (Harvard School of Public Health, USA) for the generous supply of macrophage cell line. The work was supported by international scholar funding from the Ministry of Education of Japan and a grant for medical research from Tianjin University of Traditional Chinese Medicine. References [1] A. Tluczek, T. Becker, A. Laxova, A. Grieve, C.N. Racine Gilles, M.J. Rock, W.M. Gershan, C.G. Green, P.M. Farrell, Relationships among health-related quality of life, pulmonary health, and newborn screening for cystic fibrosis, Chest 140 (1) (2011) 170–177. [2] S. Vento, F. Cainelli, Z. Temesgen, Lung infections after cancer chemotherapy, Lancet Oncol. 9 (10) (2008) 982–992. [3] S. Fujitani, H.Y. Sun, V.L. Yu, J.A. Weingarten, Pneumonia due to Pseudomonas aeruginosa: part I: epidemiology, clinical diagnosis, and source, Chest 139 (4) (2011) 909–919.

15

[4] K. Yuan, C. Huang, J. Fox, D. Laturnus, E. Carlson, B. Zhang, Q. Yin, H. Gao, M. Wu, Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages, J. Cell Sci. 125 (2) (2012) 507–515. [5] D. Dacheux, B. Toussaint, M. Richard, G. Brochier, J. Croize, I. Attree, Pseudomonas aeruginosa cystic fibrosis isolates induce rapid, type III secretion-dependent, but ExoU-independent, oncosis of macrophages and polymorphonuclear neutrophils, Infect. Immun. 68 (2000) 2916–2924. [6] C.C. Caldwell, Y. Chen, H.S. Goetzmann, Y. Hao, M.T. Borchers, D.J. Hassett, L.R. Young, D. Mavrodi, L. Thomashow, G.W. Lau, Pseudomonas aeruginosa exotoxin pyocyanin causes cystic fibrosis airway pathogenesis, Am. J. Pathol. 175 (6) (2009) 2473–2488. [7] T.S. Wilkinson, K. Dhaliwal, T.W. Hamilton, A.F. Lipka, L. Farrell, D.J. Davidson, R. Duffin, A.C. Morris, C. Haslett, J.R. Govan, C.D. Gregory, J.M. Sallenave, A.J. Simpson, Trappin-2 promotes early clearance of Pseudomonas aeruginosa through CD14-dependent macrophage activation and neutrophil recruitment, Am. J. Pathol. 174 (4) (2009) 1338–1346. [8] E.E. Nieuwenhuis, T. Matsumoto, M. Exley, R.A. Schleipman, J. Glickman, D.T. Bailey, N. Corazza, S.P. Colgan, A.B. Onderdonk, R.S. Blumberg, CD1ddependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung, Nat. Med. 8 (6) (2002) 588–593. [9] D. Descamps, M. Le Gars, V. Balloy, D. Barbier, S. Maschalidi, M. Tohme, M. Chignard, R. Ramphal, B. Manoury, J.M. Sallenave, Toll-like receptor 5 (TLR5), IL-1b secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing, Proc. Natl. Acad. Sci. USA 109 (5) (2012) 1619–1624. [10] A.R. Hauser, The type III secretion system of Pseudomonas aeruginosa: infection by injection, Nat. Rev. Microbiol. 7 (9) (2009) 654–665. [11] F.S. Sutterwala, L.A. Mijares, L. Li, Y. Ogura, B.I. Kazmierczak, R.A. Flavell, Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome, J. Exp. Med. 204 (13) (2007) 3235–3245. [12] T. Yamada, M. Goto, V. Punj, O. Zaborina, K. Kimbara, T.K. Das Gupta, A.M. Chakrabarty, The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53, Infect. Immun. 70 (12) (2002) 7054–7062. [13] J. Zhang, R. Jiang, S. Tagayama, Y. Tanaka, Survival of virulent Mycobacterium tuberculosis involves preventing apoptosis induced by Bcl-2 upregulation and release resulting from necrosis in J774 macrophages, Microbiol. Immunol. 49 (9) (2005) 845–852. [14] J. Zhang, H. Takayama, Y. Matsuba, R. Jiang, Y. Tanaka, Induction of apoptosis in macrophage cell line, J774, by the cell-free supernatant from Pseudomonas aeruginosa, Microbiol. Immunol. 47 (3) (2003) 199–206. [15] P. Vandenabeele, L. Galluzzi, T. Vanden Berghe, G. Kroemer, Molecular mechanisms of necroptosis: an ordered cellular explosion, Nat. Rev. Mol. Cell Biol. 11 (10) (2010) 700–714. [16] G.N. Schroeder, N.J. Jann, H. Hilbi, Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death, Microbiology 153 (Pt 9) (2007) 2862–2876. [17] E.A. Miao, I.A. Leaf, P.M. Treuting, D.P. Mao, M. Dors, A. Sarkar, S.E. Warren, M.D. Wewers, A. Aderem, Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria, Nat. Immunol. 11 (12) (2010) 1136–1142. [18] S. Lilo, Y. Zheng, J.B. Bliska, Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ, Infect. Immun. 76 (9) (2008) 3911–3923. [19] M. Mueller, U. Grauschopf, T. Maier, R. Glockshuber, N. Ban, The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism, Nature 459 (7247) (2009) 726–730. [20] M. Divangahi, D. Desjardins, C. Nunes-Alves, H.G. Remold, S.M. Behar, Eicosanoid pathways regulate adaptive immunity to Mycobacterium tuberculosis, Nat. Immunol. 11 (8) (2010) 751–758. [21] J.H. Wang, Y.J. Zhou, P. He, Staphylococcus aureus induces apoptosis of human monocytic U937 cells via NF-kappaB signaling pathways, Microb. Pathog. 49 (5) (2010) 252–259. [22] L. Chávez-Galán, I. Sada-Ovalle, R. Baez-Saldaña, R. Chávez, R. Lascurain, Monocytes from tuberculosis patients that exhibit cleaved caspase 9 and denaturalized cytochrome c are more susceptible to death mediated by Tolllike receptor 2, Immunology 135 (4) (2012) 299–311. [23] L.E. Bermudez, A. Parker, M. Petrofsky, Apoptosis of Mycobacterium aviuminfected macrophages is mediated by both tumour necrosis factor (TNF) and Fas, and involves the activation of caspases, Clin. Exp. Immunol. 116 (1) (1999) 94–99. [24] H. Sohn, J.S. Kim, S.J. Shin, K. Kim, C.J. Won, W.S. Kim, K.N. Min, H.G. Choi, J.C. Lee, J.K. Park, H.J. Kim, Targeting of Mycobacterium tuberculosis heparinbinding hemagglutinin to mitochondria in macrophages, PLoS Pathog. 7 (12) (2011) e1002435. [25] D.C.A. Gray, S. Mahrus, J.A. Wells, Activation of specific apoptotic caspases with an engineered small-molecule-activated protease, Cell 142 (4) (2010) 637– 646. [26] H. Ashida, H. Mimuro, M. Ogawa, T. Kobayashi, T. Sanada, M. Kim, C. Sasakawa, Cell death and infection: a double-edged sword for host and pathogen survival, J. Cell Biol. 195 (6) (2011) 931–942. [27] Y. Zhang, X. Li, A. Carpinteiro, E. Gulbins, Acid sphingomyelinase amplifies redox signaling in Pseudomonas aeruginosa-induced macrophage apoptosis, J. Immunol. 181 (6) (2008) 4247–4254.