MAJOR ARTICLE
Stem Bromelain–Induced Macrophage
Apoptosis and Activation Curtail
Mycobacterium tuberculosis Persistence
Sahil Mahajan, Vemika Chandra, Sandeep Dave, Ravikanth Nanduri, and Pawan Gupta
Institute of Microbial Technology, Chandigarh, India
Background. Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to
usurp its host’s innate immune response, killing millions of infected people annually. One approach to manage
infection is prevention through the use of natural agents. In this regard, stem bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a
remarkable ability to induce the innate and acquired immune systems, is important.
Methods. We evaluated SBM’s ability to induce apoptosis and free-radical generation in macrophages. We
also studied antimycobacterial properties of SBM and its effect on foamy macrophages.
Results. SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and
downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels
of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and
nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection.
These conditions created an environment for the increased clearance of M. tuberculosis.
Conclusions. Together these data provide a mechanism for antimycobacterial activity of SBM and provide
important insights for the use of cysteine proteases as immunomodulatory agents.
Infection with Mycobacterium tuberculosis affects
about one-third of the human population and is responsible for millions of deaths worldwide [1]. In
2008, there were an estimated 8.9–9.9 million incident
cases of tuberculosis, 9.6–13.3 million prevalent cases
of tuberculosis, and 1.1–1.7 million deaths from tuberculosis (Global tuberculosis control: a short update to
the 2009 report). Although antimycobacterial therapy
exists, the longer duration of the therapy and the
failure in the compliance of treatment leads to the
emergence of multidrug-resistant tuberculosis and
extensively drug-resistant tuberculosis [2, 3]. Thus, developing new strategies to combat M. tuberculosis has
Received 11 August 2011; accepted 18 January 2012; electronically published
21 May 2012.
Correspondence: Pawan Gupta, Institute of Microbial Technology, Protein
Science and Engineering, Sector 39A, Chandigarh, Punjab 160036, India ( pawan@
imtech.res.in).
The Journal of Infectious Diseases 2012;206:366–76
© The Author 2012. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected].
DOI: 10.1093/infdis/jis354
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become an important goal. In earlier times, shifting
the affected individuals to sanatoriums at higher
altitudes and vitamin D therapy were used to treat
human tuberculosis [4–7]. Currently, several naturally
occurring plant products for the treatment of tuberculosis are being evaluated [8–11]. Some of these are
showing promise in tuberculosis patients.
Stem bromelain (SBM) (3.4.22.32), a widely used
phytotherapeutic member of the sulfhydryl proteolytic
enzyme family, is obtained from Ananas comosus and
is of significant clinical interest [12]. Many studies
have demonstrated bromelain’s ability to treat
immune-mediated conditions including reduction of
thrombogenesis, hypertension, platelet aggregation,
angina pectoris, and surgical trauma [13]. Bromelain
treatment of T cells has been shown to markedly
increase T-cell activation via CD2 and enhance
antigen-independent T-cell binding to monocytes
[14]. Bromelain also activates various innate immune
cells including macrophages, dendritic cells, and
natural killer cells, and this activation is independent
of endotoxin receptors [15, 16]. Earlier reports have
highlighted the proapoptotic properties of bromelain
Q1
in mouse skin tumors and breast cancer cells [17]. Apoptosis
is also a strategy used by the host immune system against intracellular pathogens. Mycobacterium tuberculosis proliferates
in macrophages by preventing them from undergoing apoptosis [18]. Apoptosis of infected macrophages results in the formation of apoptotic bodies that sequester bacilli and are later
cleared from the host cell by phagocytes [19]. Moreover, reactive oxygen species (ROS) and reactive nitrogen intermediates
(RNI), both of which possess bactericidal activity, are key in
the clearance of M. tuberculosis from the infected macrophages [20, 21]. A characteristic trait of mycobacterial infection is
the formation of foamy (lipid laden) macrophages [22]. The
lipid bodies in the macrophages harbor nonreplicating M. tuberculosis, providing them with a source of carbon and supporting their survival [23].
In this study, we show that SBM modulates apoptosis in
peritoneal macrophages by shifting the balance in favor of
proapoptotic proteins. These events are also associated with
the increase of various ROS and RNI. Furthermore, SBM
treatment upregulates surface expression of costimulatory
molecules and increases the production of proinflammatory
cytokines. We also show that SBM treatment decreases the
bug burden of M. tuberculosis in primary murine and human
macrophages. Finally, SBM treatment decreases the foamy
phenotype in infected macrophages; one of the reasons for
this decrease is SBM’s ability to cleave the extracellular
domain of CD36, the molecule that accounts for most of the
oxidized low-density lipoprotein uptake in macrophages.
MATERIALS AND METHODS
Reagents
Dulbecco’s modified Eagle’s medium, fetal bovine serum,
LysoTracker Red, and CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were purchased from Invitrogen
Life Technologies. Stem bromelain, Ampliflu Red, dihydrorhodamine 123, nitro blue tetrazolium (NBT), and Oil Red O
were from Sigma. Anti-Bcl-2, anti-caspase 3, anti-caspase 9,
cleaved PARP antibodies, and anti-Bax antibodies were from
Cell Signaling Technology. Allophycocyanin (apc)–conjugated
CD80 and CD86 and all enzyme-linked immunosorbent assay
(ELISA) reagents were purchased from BD Biosciences, and
the TUNEL assay kit was procured from Calbiochem. All
other standard reagents were procured from Sigma unless otherwise mentioned.
BALB/c mice were immunized by intraperitoneal injection
with 4% thioglycollate medium, and elicited peritoneal macrophages were harvested 4–5 days later. Mouse alveolar macrophages were harvested by bronchoalveolar lavage (BAL). BAL
was performed in prewarmed phosphate-buffered saline
(PBS). Cells from the BAL fluid were collected by centrifugation at 800 × rpm for 5 minutes at 4°C.
Primary Macrophage Differentiation
Human peripheral blood mononuclear cells (PBMCs) were
derived from fresh blood drawn from healthy individuals by
Ficoll-Hypaque density gradient centrifugation. PBMCs were
then allowed to differentiate to macrophages over a period of
7 days in complete RPMI-1640 medium along with 50 ng/mL
granulocyte-macrophage colony-stimulating factor.
Ethics Statement
All experiments were approved by the Institutional Animal
Ethics Committee of the Institute of Microbial Technology
and performed according to the National Regulatory Guideline issued by the Committee for the Purpose of Supervision
of Experiments on Animals (No. 55/1999/CPCSEA), Ministry
of Environment and Forest, government of India. The study
on human subjects was conducted strictly in accordance with
the ethical guidelines for biomedical research on human subjects by the Central Ethics Committee on Human Research,
ICMR–2000 and those as contained in the Declaration of
Helsinki. Each participant was provided with written information about the study and written consent was obtained.
Ampliflu Red Assay to Measure Hydrogen Peroxide
For hydrogen peroxide measurement, peritoneal macrophages
were stimulated for 12 hours at 37°C with 50 µg/mL of SBM.
The cells were washed 3 times with PBS and were incubated
with the hydrogen peroxide–specific fluorescence probe
Ampliflu Red (10 µM) along with 0.5 unit of horseradish
peroxidase (HRP) for 30 minutes at 37°C. The supernatant
was collected and fluorescence was measured using excitation/
emission wavelengths of 563/587 nm.
Dihydrorhodamine 123 Assay to Measure Peroxynitrite
Peritoneal macrophages were stimulated for 12 hours at 37°C
with 50 µg/mL SBM. The cells were washed 3 times with PBS
followed by incubation with 10 µm dihydrorhodamine (DHR)
123 probe for 30 minutes at 37°C. The cells were harvested
and subjected to fluorescence-activated cell sorting (FACS) to
determine the levels of peroxynitrite.
Isolation of Peritoneal Macrophages and Alveolar
Macrophages
Nitro Blue Tetrazolium Assay to Measure Superoxide
Male inbred BALB/c mice were obtained at 6–8 weeks of age
from the Institute of Microbial Technology (IMTECH) animal
house facility. Only those mice that displayed no sign of pathological manifestation were used for macrophage collection.
Peritoneal macrophages were stimulated as described above
and incubated with NBT (1 mg/mL) for 1 hour. The macrophages were washed 3 times with PBS to remove extracellular
NBT. The NBT deposited inside the cells was then dissolved
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using 2 M potassium hydroxide followed by the addition of
dimethyl sulfoxide. The solution was collected and the absorbance was recorded at 560 nm.
Griess Assay to Measure Nitrite
Peritoneal macrophages were stimulated as described above. To
determine nitrite accumulation, an indicator of nitric oxide (NO)
production, equal volumes (50 µL) of supernatants and Griess
reagent (Sigma) were mixed for 10 minutes. Absorbance at 540
nm was measured and compared to a standard nitrite curve.
CMFDA Assay to Measure Free Glutathione
Macrophages were treated for 12 hours with 50 µg/mL of SBM
at 37°C. Cells were washed 3 times with PBS and incubated
with CMFDA for 30 minutes. Any extracellular CMFDA was
removed by washing the cells 3 times with PBS. The cells were
further incubated for 30 minutes and the fluorescence images
of the CMFDA bound cells were taken. Next, fluorescence intensities of the images were calculated using Adobe Photoshop
7.0 software.
TUNEL Assay
Cells were treated with vehicle or 50 µg/mL of SBM for 12
hours followed by 3 washes with tris buffer saline. The
TUNEL assay was performed using a Fluorescein FRagEL
DNA Fragmentation Detection Kit (Calbiochem) according to
the manufacturer’s protocol.
Surface Staining of CD80 and CD86
Peritoneal macrophages (5 × 105 per well) were treated with
SBM (50 µg/mL) for 12 hours. Cells were then detached from
the wells and stained with apc anti-mouse CD80 and CD86
for 30 minutes at 4°C. Finally, cells were washed and fixed in
1% paraformaldehyde. The flow cytometry data were acquired
and analyzed using FACSCalibur.
Enzyme-Linked Immunosorbent Assay
Murine interleukin IL 12p70 (IL-12p70), interleukin 10 (IL-10),
tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in
the culture supernatant of peritoneal macrophages treated with
SBM (10–50 µg/mL) were quantified by ELISA (BD OptEIA,
BD Biosciences) according to the manufacturer’s instructions.
Enumeration of Intracellular Bacteria
Primary murine alveolar macrophages, and peritoneal macrophages or primary human macrophages were incubated with
SBM for 12 hours followed by infection of H37Ra or H37Rv
at 1:5 multiplicity of infection (MOI) for 4 hours. The cells
were then washed with media to remove any unphagocytosed
bacteria and kept for another 6 hours. At this point the cells
were solubilized, and the number of colony-forming units
(CFUs) was determined using a 1:10 dilution of lysate.
Western Blot Analysis
Lipid Body Staining
Supernatant from untreated and SBM-treated macrophages was
collected, and the supernatant proteins were separated on a 10%
sodium dodecyl sulfate (SDS) gel. Bands that appeared in the
supernatant of only the SBM-treated cells were subjected to
mass spectrometry analysis. Gel slices of band D were subjected
to overnight in-gel tryptic digestion. The samples were analyzed
by MALDI-TOF MS (Voyager, Applied Biosystems) using
α-cyano-4-hydroxycinnamic acid as a matrix in a positive ion
reflection mode. The peptide mass fingerprinting data from 5
experiments were searched via a MASCOT (www.matrixscience.
com) PMF data search. The sequence coverage is shown from
the result of PMF.
Cells were infected as described above. The cells were fixed
with 4% paraformaldehyde for 5 minutes, stained with 0.5%
Oil Red O for 30 minutes at room temperature, and incubated
with hematoxylin to counterstain the nucleus. Cells were
rinsed with PBS, mounted on glass slides, and imaged. Lipid
bodies were counted in 50 consecutive macrophages per slide
using a light microscope with a ×100 objective lens.
Confocal Microscopy
Peritoneal macrophages were seeded onto 16-mm diameter
glass coverslips pretreated with poly-lysine in 12-well tissue
culture plates at a density of 5 × 105 cells per coverslip. Cells
were treated with SBM for 12 hours, followed by 4 hours of
infection with GFP-H37Ra at 1:5 MOI. Cells were then
washed with medium to remove any unphagocytosed bacteria
and kept for an additional 4 hours. Cells were then incubated
with 100 nM LysoTracker for 2 hours at 37°C and fixed with
4% paraformaldehyde. The coverslips were washed thoroughly
with PBS and mounted on slides with antifade. The stained
cells were observed with an LSM 510 Meta Carl Zeiss confocal
microscope.
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Cells were treated as described above and Western blot analyses were performed as described previously [12]. Membranes
were incubated overnight with primary antibodies (1:3000
dilution) followed by incubation with HRP-conjugated
secondary antibodies (1:10 000 dilution) and were visualized
using Luminata Forte Western HRP substrate (Millipore).
Mass Spectrometric Analysis
RESULTS
SBM Causes Apoptosis in Peritoneal Macrophages
To assess whether SBM mediates induction of apoptosis, we
first evaluated the effect of SBM on the cleavage of PARP.
Immunoblot analysis showed that full-size PARP protein was
cleaved to yield an 89-kD fragment after treatment of cells
for 12 hours with SBM alone (10–50 µg/mL) or with SBM
(50 µg/mL) and lipopolysaccharide (LPS) (1 µg/mL) together.
Figure 1. Stem bromelain (SBM) treatment induces apoptosis in macrophages by modulating apoptotic proteins. Effect of SBM treatment of peritoneal macrophages on (A) cleavage of PARP, (B) cleavage of caspase 3 and caspase 9, and (C) expression of Bax and Bcl-2. The cells were treated with
vehicle or specified concentrations of lipopolysaccharide (LPS) or SBM for 12 h and harvested. Cell lysates were prepared and Western blots were
probed with specified antibodies. β-actin served as a loading control. D, Peritoneal macrophages were treated with vehicle or 50 µg/mL of SBM and
subjected to a TUNEL assay. TUNEL-positive cells were visualized by fluorescein-conjugated deoxynucleotides (green) and the nucleus by 4,6-diamidino2-phenylindole (DAPI; blue). Data are representative of 3 independent experiments with similar results.
No cleavage was observed in untreated cells or in cells that were
treated with LPS alone (Figure 1A). A similar pattern was seen
with caspase 3 and caspase 9; fragments of cleaved caspase 3
and cleaved caspase 9 appeared in cells treated with SBM alone
and in cells treated with SBM and LPS together (Figure 1B).
Because Bax and Bcl-2 play crucial roles in apoptosis, we next
studied the effects of SBM on their protein levels. An increase in
the expression of Bax and a significant decrease in the expression of Bcl-2 were evident after SBM treatment (Figure 1C). A
TUNEL assay at 50 µg/mL of SBM revealed that the number of
TUNEL-positive cells in SBM-treated cells was more than the
cells that received no treatment (Figure 1D). These results confirmed the induction of apoptosis by SBM in macrophages.
SBM treatment also induces apoptosis in H37Rv-infected macrophages (Supplementary Figure 1).
CD86, mouse peritoneal macrophages were treated for 12
hours with medium alone (control) or 50 µg/mL SBM, and
the expression of these molecules was determined by FACS
(Figure 2A and 2B). Intriguingly, an increased expression of
costimulatory molecules after SBM treatment compared with
that of the control cells was observed. We next evaluated the
effect of SBM treatment on the secretion of cytokines. Stimulation of peritoneal macrophages with SBM (10–50 µg/mL) resulted in the dose-dependent secretion of IL-12p70, TNF-α,
and IL-6 that was significantly higher than that observed in
unstimulated cells at all doses (Figure 2C–E). SBM treatment
slightly curtailed the secretion of IL-10 at all tested doses
(Figure 2F). Overall, these results indicate SBM’s ability to
enhance the activation of macrophages.
ROS and RNI Responses to SBM
SBM Modulates Costimulatory Molecules and Inflammatory
Cytokines
To determine whether pretreatment with SBM affects the
surface expression of costimulatory molecules CD80 and
We next inquired whether SBM stimulation augmented the
production of various ROS and RNI in macrophages.
Cysteine proteases have been demonstrated to induce ROS
in antigen-presenting cells (APCs) and NO in macrophages
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Figure 2. Stem bromelain (SBM) treatment increases the expression of CD80, CD86, and proinflammatory cytokines and decreases the expression of
interleukin 10 (IL-10). A and B, Cells were treated for 12 h with vehicle or 50 µg/mL SBM, stained for the surface expression of CD80 and CD86, and
analyzed by flow cytometry. Shaded histograms represent untreated macrophages, and open histograms represent SBM-treated macrophages. B–E, Cells
were treated for 12 h with vehicle or various concentrations of SBM (10–50 µg/mL), the conditioned media were collected, and (B) interleukin 12p70
(IL-12p70), (C) tumor necrosis factor (TNF) α, (D) interleukin 6 (IL-6), and (E) interleukin 10 (IL-10) were quantified by enzyme-linked immunosorbent
assay. Values represent the mean ± SD of 3 independent experiments performed in triplicate.
[15, 24]. Free glutathione (GSH) quenches intracellular ROS
and provides guard against ROS-mediated damage. So, we
first looked at the levels of free GSH on SBM treatment
using the CMFDA fluorescence probe. Clearly, SBM treatment decreases the level of GSH as indicated by the low
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fluorescence intensity (Figure 3A). We next investigated the
role of SBM in the generation of superoxide and hydrogen
peroxide species. Peritoneal macrophages were treated with
SBM (50 µg/mL) or LPS (1 µg/mL) or with SBM and LPS
combined. Although LPS is known to induce optimal levels
Figure 3. The levels of glutathione (GSH) were decreased and those of superoxide anion, hydrogen peroxide (H2O2), nitric oxide (NO), and peroxynitrite
(ONOO_) was increased on stem bromelain (SBM) treatment. A, Macrophages were treated with vehicle or SBM (50 µg/mL) and the levels of GSH were
determined by using 5-chloromethylfluorescein diacetate (CMFDA) fluorescent probe. Macrophages were stimulated for 12 h with 1 µg/mL of lipopolysaccharide (LPS), 50 µg/mL of SBM, or LPS and SBM together. B, Superoxide production was measured by incubating the cells with nitro blue tetrazolium
for 1 h. C, H2O2 generation was measured using Ampliflu Red fluorescence probe by incubating the cells for 30 min D, Griess assay was used to measure
nitrite, an indicator of NO production. Equal volumes of supernatant and Griess reagent were mixed for 10 min, and the absorbance was measured. E,
ONOO_ generation was measured using DHR123 fluorescence probe. Cells were harvested after incubation with DHR123 for 30 min and analyzed by
fluorescence-activated cell sorting. Values represent the mean ± SD of 3 independent experiments performed in triplicate. *P < .05; **P < .01.
of superoxide and hydrogen peroxide in peritoneal macrophages [15, 16], the role of SBM in this context is unknown.
In our experiments, SBM treatment promoted superoxide
generation in peritoneal macrophages by 42%, whereas the
level of hydrogen peroxide was elevated by 22%. LPS also
brought a significant change in the levels of both ROI
species, but the increase was comparatively less than with
SBM (Figure 3B and 3C).
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Figure 4. Stem bromelain (SBM) modulates intracellular H37Ra and H37Rv survival. SBM-treated murine peritoneal macrophages were infected with
(A) H37Ra or (B) H37Rv or SBM-treated murine alveolar macrophages, and primary human macrophages (MΦ) were infected with (C and D) H37Rv; the
pathogen survival was assessed by counting colony-forming units (CFUs). CFU counts are plotted as the mean ± SD of 3 independent experiments.
*P < .05; **P < .01. E, Evaluation of H37Ra survival by confocal microscopy. The extent of colocalization of H37Ra-GFP with lysosomes was determined
in macrophages treated with vehicle or SBM. Images shown in each group are those obtained for differential interference contrast (DIC), H37Ra-GFP,
lysosomes (LysoTracker), and the merged images of H37Ra-GFP and lysosomes. Images are representative of 3 individual experiments. Abbreviation:
PBMC, peripheral blood mononuclear cell.
We also monitored the effect of SBM on RNI and NO.
SBM and LPS each induced significant levels of NO, although
the induction was higher in LPS-stimulated cells (Figure 3D).
Treatment of the macrophages with SBM and LPS together
had no significant effect on NO production compared to treatment with LPS alone (Figure 3D). These data suggest that
SBM is a potent inducer of NO and that there is no synergism
between SBM and LPS in the production of NO. Peroxynitrite
levels in the cells were measured after incubating them with
DHR123. Both LPS and SBM induced the production of peroxynitrite, but the induction was higher in SBM-treated cells
(Figure 3E). H37Rv-infected macrophages also showed an increase in the generation of free radicals when treated with
SBM (Supplementary Figure 2).
SBM-Treated Macrophages Showed Increased Killing of
H37Ra and H37Rv
We next inquired whether SBM stimulation of macrophages
plays any role in increased killing of M. tuberculosis. SBM
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treatment of macrophages generated free radicals, and freeradical generation by activated murine macrophages has been
shown to provide successful clearance of virulent M. tuberculosis [21]. To assess whether SBM treatment of macrophages
improves clearance of M. tuberculosis, we infected peritoneal
macrophages with H37Ra and H37Rv at 1:5 MOI. Clearly, the
treated macrophages showed better clearance of bacteria; the
bacterial burden in untreated macrophages remained very
high as measured in CFUs (Figure 4A and 4B). We also observed increased clearance of H37Rv in SBM-treated murine
alveolar macrophages and primary human macrophages
(Figure 4C and 4D). Next we performed confocal microscopy
to determine if the results obtained in CFUs correlated with
the extent to which M. tuberculosis colocalizes with the lysosomes in infected macrophages. In untreated cells, the extent
of H37Ra colocalization with the lysosomes was far less than
the colocalization observed in SBM-treated macrophages
(Figure 4E). Thus, these results clearly confirm the antimycobacterial activity of SBM.
Figure 5. Stem bromelain (SBM) modulates the foamy phenotype by cleaving CD36. A, Oil Red O staining was performed to evaluate lipid biogenesis
in vehicle-treated, SBM-treated, H37Ra-infected, and SBM-treated/H37Ra-infected macrophages. Lipid bodies were visualized by light microscopy with
a ×100 objective lens and counted in 50 consecutive macrophages on each side. B, Oil Red O staining was performed to evaluate lipid biogenesis in
vehicle-treated, SBM-treated, H37Rv-infected, and SBM-treated/H37Rv-infected macrophages. Lipid bodies were enumerated as mentioned above.
*P < .05; **P < .01. C, Protein bands that appeared in the supernatant of SBM-treated cells were separated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis and subjected to mass spectrometry. The matches for the identified protein and the peptide coverage analysis are shown in the
figure.
SBM Cleaves CD36 and Decreases Foamy Phenotype in
Infected Macrophages
We next addressed the role of SBM in the formation of foamy
macrophages, a nutrient-rich reservoir that contributes to
M. tuberculosis persistence. More lipid bodies, characteristic of
foamy phenotype, were observed upon macrophage infection
with H37Ra and H37Rv (Figure 5A and 5B). This foamy phenotype was drastically reduced in infected macrophages
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treated with SBM. Intriguingly, we also observed that SBM decreases the number of lipid bodies in uninfected macrophages
(Figure 5A and 5B). We suspected that this decrease was
because of the proteolytic ability of SBM that it exerts on the
surface molecules of macrophages. In an attempt to identify
surface molecules that are affected by SBM treatment, we collected the supernatant from untreated and SBM-treated macrophages and separated the supernatant proteins on a 10%
SDS gel. Bands that appeared in the supernatant of only the
SBM-treated cells were subjected to mass spectrometry analysis. Among them we repeatedly identified CD36, a surface
molecule that accounts for most of the oxidized LDL uptake
(Figure 5C). These results suggest that SBM treatment decreases lipid body formation in both uninfected and infected macrophages by cleaving the surface receptor CD36.
DISCUSSION
In the last couple of decades, the use of plant-derived compounds to manage M. tuberculosis has gained considerable interest. A variety of products obtained from various plant
species are used in traditional methods of medicine to alleviate
the symptoms of tuberculosis, although not much emphasis
had been placed on understanding their anti–M. tuberculosis
properties [8–11]. Recently, extracellular proteases have been
shown to have immunomodulatory properties and have been
implicated in both innate and adaptive immunity [14, 15, 25–29].
In this study, we investigated the antimycobacterial role of
stem bromelain, a extracellular cysteine protease obtained
from Ananas comosus, and these results provide at least a
partial mechanism for how SBM alleviates the pathogenic
burden of M. tuberculosis.
The principal cell that harbors M. tuberculosis inside the body
is the macrophage. With avirulent strains of M. tuberculosis,
macrophages undergo apoptosis; with virulent strains, they
undergo necrosis [30–33]. Whereas apoptosis prevents the
spread of infection by sequestering the bacteria inside apoptotic
bodies, necrotic cell death releases the bacteria, which are then
free to infect neighboring cells. This advocates that apoptosis of
the infected macrophages abolish a growth niche for M. tuberculosis. In this regard, SBM’s proapoptotic properties are potentially important. Bromelain has been earlier shown to induce
apoptosis by reducing NF-κB–driven COX-2 expression in
DMBA-TPA–induced mouse skin tumors [34]. MCF-7 cells
when exposed to bromelain showed induction of autophagy followed by cell death, and the use of 3-methyladenine, an autophagic inhibitor, reduced the bromelain-induced autophagic
and apoptotic population [35]. The exact mechanism of SBMinduced apoptosis is not known, but its ability to suppress
ERK1/2 activation is thought to be an important for its proapoptotic properties [17, 34, 35]. In this study, SBM’s proapoptotic activity in macrophages was confirmed by the presence of
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cleaved PARP, cleaved caspase 3, and cleaved caspase 9 and by
the appearance of TUNEL-positive cells (Figure 1A, 1B, and
1D). Bcl-2 and related members of the Bcl-2 family are important regulators of apoptotic pathways [36]. Aberrant elevation of
Bcl-2 is observed in H37Rv-infected macrophages and enables
infected macrophages to avoid apoptotic elimination [37]. Bcl-2
also has the ability to neutralize Bax proapoptotic effects [38].
Thus the ratio of proapoptotic and antiapoptotic factors determines whether the cell lives or dies. In our study, SBM treatment
caused a decrease in Bcl-2 expression and an increase in protein
expression of Bax, and this shift in favor of proapoptotic proteins sets up the cell for apoptosis.
Bromelain’s immunomodulatory effects are well documented and have been seen on almost all immune cells. Bromelain
and papain stimulate the production of TNF-α, interleukin 1β
(IL-1β), and IL-6 from human PBMCs in a time- and dosedependent manner [39, 40]. It has also been reported that bromelain increases interferon (IFN)-γ–dependent TNF-α, IL-1β,
and IL-6 production by human PBMCs and IFN-γ–mediated
NO and TNF-α production by macrophages [15, 27, 41]. Bromelain also induces the secretion of IL-6 from modified mixed
lymphocyte culture in a dose-dependent manner [27]. Secretion of proinflammatory cytokines constitutes an important
component of the host defense [42]. Many plant-derived molecules are capable of modulating redox potential, and it is a
characteristic, early event in the progression of cells toward
apoptosis [43]. Previous reports suggest that bromelain
induced ROS production in monocytes of patients suffering
from X-linked agammaglobulinemia and in carcinoma A431
and melanoma A375 cells [17, 44]. Our results suggest that
SBM treatment generates oxidative stress by increasing ROS
and reactive nitrogen species in macrophages.
Our results demonstrate for the first time the antimicrobial
activity of SBM. Pretreatment of macrophages with bromelain
impeded M. tuberculosis survival and decreased the formation
of foamy macrophages. Recently, it has been suggested that lipid
droplets provide a safe niche for the survival of nonreplicating
M. tuberculosis and that mycobacteria use host lipids as their
carbon source [23, 45]. Also, HOC-12.5 (macrocyclon) and
HOC-60, which respectively induce and prevent lipid droplets
inside macrophages, have been shown to enhance and inhibit
M. tuberculosis growth, respectively [46]. The role of oxidized
LDL in formation of foam cells is well documented, and CD36
is a major receptor in the oxidized LDL uptake in macrophages
[47, 48]. Our results indicate that SBM cleaves the extracellular
fragment of CD36; this proteolytic cleavage accounts for the decreased lipid burden in macrophages. Further investigation of
the mechanism of SBM in curtailing macrophage lipids will help
illuminate its potential for use in tuberculosis patients.
Based on the present findings, it is attractive to suggest the
use of SBM in antimycobacterial therapy along with the frontline drugs currently used for M. tuberculosis patients.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://www.oxfordjournals.org/our_journals/jid/). Supplementary
materials consist of data provided by the author that are published to
benefit the reader. The posted materials are not copyedited. The contents
of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes
Acknowledgments. We are grateful to Deepak Bhatt and Anjali
Koundal for their support on confocal microscopy; Dr Girish Sahni, Dr
Shweta Jain, Ankita Saini, and Kitdorlang H. Dkhar for their effort and
help; and IMTECH (a constituent laboratory of Council of Scientific and
Industrial Research) for support and use of its facilities.
Financial support. This work was supported by Department of
Biotechnology, (grant numbers BT/PR11538/BRB and BT/01/IYBA/2009),
government of India; the Council of Scientific and Industrial Research (fellowships to S. M., V. C., and S. D.); and the University Grants Commission
(fellowship to R. K. N.).
Author contributions. S. M. and P. G. designed research; S. M., V. C.,
S. D., and R. K. N. performed research; S. M., V. C., and P. G. analyzed
data; and S. M. and P. G. wrote the paper.
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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