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RESEARCH ARTICLE
Virulent Mycobacterium bovis Beijing Strain
Activates the NLRP7 Inflammasome in THP-1
Macrophages
Yang Zhou
1
, Syed Zahid Ali Shah
1
, Lifeng Yang
1
, Zhongqiu Zhang
2
, Xiangmei Zhou
1
*,
Deming Zhao
1
*
1National Animal Transmissible Spongiform Encephalopathy Laboratory, Key Laboratory of Animal
Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key
Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China, 2Veterinary Bureau,
Ministry of Agriculture of the People’s Republic of China, Beijing 100125, China
*zhouxm@cau.edu.cn (XZ); zhaodm@cau.edu.cn (DZ)
Abstract
Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals,
including humans. Macrophages are the first line of host defense. They secrete proinflam-
matory cytokines, such as interleukin-1 beta (IL-1β), in response to mycobacterial infection,
but the underlying mechanisms by which human macrophages are activated and release
IL-1βfollowing M.bovis infection are poorly understood. Here we show that the ‘nucleotide
binding and oligomerization of domain-like receptor (NLR) family pyrin domain containing 7
protein’(NLRP7) inflammasome is involved in IL-1βsecretion and caspase-1 activation
induced by M.bovis infection in THP-1 macrophages. NLRP7 inflammasome activation pro-
motes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha
(TNF-α), Chemokine (C-C motif) ligand 3 (CCL3) and IL-1βmRNAs. Thus, the NLRP7
inflammasome contributes to IL-1βsecretion and induction of pyroptosis in response to M.
bovis infection in THP-1 macrophages.
Introduction
Mycobacterium bovis, a member of the M.tuberculosis complex, is the etiological agent of
bovine tuberculosis which is estimated to infect more than 50 million cattle per annum with
concomitant economic losses of approximately $3 billion worldwide [1]. M.bovis is also
responsible for a proportion of human tuberculosis (TB) cases, and can be transmitted from
human to human. About 2.8% of all human TB cases in Africa are caused by M.bovis, and
human M.bovis infection accounts for 7.6% of cases in Mexico which also contributes to the
disease incidence in the United States, although overall incidence in the Americas is low [2].
This huge zoonotic risk imposes limitations on the potential for control [3,4].
Macrophages are considered the first line of host defense against invasive microbes. Upon
infection, they initiate inflammatory responses by releasing cytokines and chemokines, such as
IL-1β, IL-18, TNF-α, and CCL3. Among these, IL-1βis a potent mediator of antimicrobial
PLOS ONE | DOI:10.1371/journal.pone.0152853 April 4, 2016 1/13
a11111
OPEN ACCESS
Citation: Zhou Y, Shah SZA, Yang L, Zhang Z, Zhou
X, Zhao D (2016) Virulent Mycobacterium bovis
Beijing Strain Activates the NLRP7 Inflammasome in
THP-1 Macrophages. PLoS ONE 11(4): e0152853.
doi:10.1371/journal.pone.0152853
Editor: Volker Briken, University of Maryland,
UNITED STATES
Received: December 31, 2015
Accepted: March 21, 2016
Published: April 4, 2016
Copyright: © 2016 Zhou et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was funded by MoSTRCUK
international cooperation project (Project No.
2013DFG32500) http://www.istcp.org.cn, National
Natural Science Foundation of China (Project No.
31572487) https://isisn.nsfc.gov.cn, Funding of State
Key Lab of Agrobiotechnology (Project No.
2012SKLAB06-14) http://cbs.cau.edu.cn, 2015 CAU
Foreign Experts Major Projects (Project No:
2012z018) http://cau.edu.cn, and High-end Foreign
Experts Recruitment Program (Project No:
GDW20151100036) http://cepms.safea.gov.cn.
responses. It contributes to the maturation of mycobacterial phagosomes into phagolysosomes,
which enhances mycobacterial elimination by macrophages. Inhibition of IL-1βactivity by
neutralizing antibody or siRNA increases intracellular mycobacterial survival [5]. Conversely,
adding exogenous IL-1βmarkedly inhibits their survival [6]. IL-1βactivity is tightly controlled
at the levels of expression, maturation, and secretion [7]. It is initially synthesized as a precur-
sor molecule, proIL-1β, in the cytosol in response to pathogen-associated molecular patterns
(PAMPs), which are sensed by evolutionarily-conserved toll-like receptors. Its maturation and
secretion requires caspase-1 activation by multiprotein complexes known as inflammasomes
[8]. The inflammasomes consist of a receptor protein, the adaptor apoptosis-associated speck-
like protein containing a caspase-activation recruitment domain (ASC), and caspase-1. Recep-
tor proteins include NLR family pyrin domain containing 3 protein (NLRP3) [9], absent in
melanoma 2 (AIM2) [10], NLR family caspase-activation recruitment domain (CARD)-con-
taining protein 4 (NLRC4) [11], and NLRP7, which is uniquely stimulated by microbial acety-
lated lipopeptides [12]. The majority of research on NLRP7 has been associated with
hydatidiform mole, an abnormal human pregnancy with hyperproliferative vesicular tropho-
blast and no fetal development [13]. A recent study showed that some live and heat killed
microbes, including Mycoplasma spp., Staphylococcus aureus and Listeria monocytogenes, acti-
vate the NLRP7 inflammasome. NLRP7 senses lipopeptides through its leucine-rich repeat
(LRR) domain [12], and results in self-oligomerization to form an inflammasome scaffold
through its nucleotide-binding and oligomerization (NACHT) domain. It interacts with ASC
via homotypic pyrin domain interactions, recruiting procaspase-1 via the CARD domain of
ASC. Procaspase-1 clustering leads to caspase-1 auto-activation and generation of active cas-
pase-1, which cleaves inactive proinflammatory cytokines into their active forms.
Little is known about the protective role of the NLRP7 inflammasome against either M.
bovis or M.tuberculosis in macrophages. M.bovis is an intracellular pathogen expressing and
secreting lipoproteins [14–16] and we demonstrate here that M.bovis infection triggers NLRP7
inflammasome activation and induction of pyroptosis in human THP-1 macrophages.
Materials and Methods
Reagents
The following antibodies and reagents were purchased from the indicated suppliers: the mouse
monoclonal antibody against NLRP7 used for immunofluorescence assay and rabbit polyclonal
anti-AIM2 antibody, Santa Cruz Biotechnology; rabbit polyclonal anti-NLRP7 antibody used
for western blotting, Pierce/Thermo Fisher Scientific; rabbit polyclonal anti-ASC antibody and
rabbit polyclonal anti-NLRP3 antibody, Sangon Biotech, Shanghai, China; goat polyclonal
anti-IL-1βantibody, R&D Systems; rabbit polyclonal anti-β-actin antibody, Proteintech,
Wuchan, China; rabbit polyclonal anti-caspase-1 antibody, ProSci Incorporated; phorbol
12-myristate-13-acetate (PMA), Sigma-Aldrich; glycine, Beijing Solarbio Science & Technol-
ogy Co., Ltd.; cytochalasin D, Cayman Chemical; and Z-YVAD-FMK, BioVision Incorporated.
THP-1 cell culture and differentiation
THP-1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and
maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal
bovine serum (FBS, Gibco). THP-1 cells were stimulated with PMA (5 ng/mL) to differentiate
into macrophages for 2 days, after which the cells were washed three times with warm phos-
phate-buffered saline (PBS). Cells were then incubated in PMA-free culture medium and rested
for a further 2 days.
Mycobacterium bovis Activates the NLRP7 Inflammasome
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Competing Interests: The authors declare that no
competing interests exist.
Bacterial culture and infection
Virulent M.bovis Beijing strain was obtained from the China Institute of Veterinary Drug Con-
trol, Beijing and grown in 7H9 Middlebrook media (BD Biosciences) supplemented with albu-
min-dextrose-catalase (ADC) enrichment solution and 0.05% Tween-80 (Difco) at 37°C. THP-
1 macrophages were infected with M.bovis at a multiplicity of infection (MOI) indicated for 2
h and then washed three times with warm PBS to remove extracellular bacteria. The samples
were harvested at the indicated time.
Small interference RNA (siRNA) transfection
THP-1 macrophages were transfected with gene-specific siRNA pools to knock down NLRP7
or ASC. Human NLRP7-targeting siRNA oligonucleotides, ASC-targeting siRNA oligonucleo-
tides and non-targeting control siRNA oligonucleotides were obtained from Shanghai Gene-
Pharma Co., Ltd (Table 1). THP-1 cells were differentiated and then incubated overnight in a
24-well plate. Prior to transfection, all medium was removed and 400 μL of fresh medium was
added. Lipofectamine 3000 transfection reagent and siRNA were added into 100 μL of serum-
free culture medium, and incubated for 10 min at room temperature. The resulting mixture
was added drop-wise onto the cells and culture medium was replaced after 24 h.
Quantitative real-time PCR
Total RNA extraction was performed using RN28-EASYspin Plus Tissue/Cell RNA Kit (Aidlab
Biotech, Beijing) and reverse transcription was performed using RevertAid First Strand cDNA
Synthesis Kit (Thermo Fisher). Quantitative PCR was carried out in a Roche LightCycler480 II
using TransStart Green qPCR SuperMix UDG (Beijing TransGen Biotech). Primers for NLRP7
were 5´-TAAGGAATGCGACTGTGAACATC-3´ forward and 5´-TGCTAACTCCGAGTCTTC
TTCT-3´ reverse. Primers for NLRP3, AIM2, and GAPDH were the same as those used in our
previous study [17].
Table 1. Sequences of ASC-targeting siRNA and non-targeting control siRNA.
Name Sequence (sense, antisense)
NLRP7-targeting siRNA:
Target Sequence 1: GACGUCACUCUGAGAAACCAATT
UUGGUUUCUCAGAGUGACGUCTT
Target Sequence 2: GUCAGAGGGUCACAUGUUATT
UAACAUGUGACCCUCUGACTT [12]
Target Sequence 3: GUGUUCCUGGAGAAUUACATT
UGUAAUUCUCCAGGAACACTT [12]
ASC-targeting siRNA:
Target Sequence 1: UCGCGAGGGUCACAAACGUTT
ACGUUUGUGACCCUCGCGATT
Target Sequence 2: UGCUGUCCAUGGACGCCUUTT
AAGGCGUCCAUGGACAGCATT
Target Sequence 3: GCAAGAUGCGGAAGCUCUUTT
AAGAGCUUCCGCAUCUUGCTT
Non-targeting siRNA: UUCUCCGAACGUGUCACGUTT
ACGUGACACGUUCGGAGAATT
doi:10.1371/journal.pone.0152853.t001
Mycobacterium bovis Activates the NLRP7 Inflammasome
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Western blotting
Cells were washed in PBS, and lysed in cold lysis buffer (Beyotime Institute of Biotechnology,
China) for 20 min. Samples were centrifuged at 12,000 ×gfor 20min and the supernatant was
boiled for 10 min after addition of loading buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 0.5%
BPB, 50% glycerol, 0.5 M DTT). For detection of IL-1βand caspase-1 released into the culture
medium, proteins were precipitated as described previously [18]. Aliquots were separated via
SDS-PAGE and the proteins were transferred to PVDF membranes (Immobilon-PSQ,
ISEQ00010, 0.2 μm). Blots were blocked by 5% non-fat milk in TBST (25 mMTris base, 137
mM sodium chloride, 2.7 mM potassium chloride and 0.05% Tween-20, pH7.4) for 1 h at
room temperature, incubated with the indicated primary antibody overnight at 4°C and the
corresponding HRP-labeled secondary antibody for 50 min at 37°C, and the signal detected
using an enhanced chemiluminescence (ECL) detection kit (Bio-Rad, USA).
Immunofluorescence
THP-1 cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Following
permeabilization with 0.1% Triton X-100 for 10 min, the cells were blocked with 1% BSA for 1
h, incubated with primary antibodies overnight at 4°C and secondary antibodies at 37°C for 1
h. Nuclei were stained with DAPI for 1 min. Finally, coverslips were mounted on slides, and
the cells were imaged using confocal microscopy. Colocalization was quantified using ImageJ
software.
Lactate dehydrogenase (LDH) release assay
LDH release was measured using LDH Cytotoxicity Assay Kit (Cayman Incorporated) accord-
ing to the manufacturer’s instructions.
Statistical analysis
All assays were performed in three independent experiments and data were analyzed using
GraphPad Prism 5.0 software and Student’s t test; p <0.05 values were considered statistically
significant.
Results
M.bovis infection induces caspase-1 activation and IL-1βsecretion in
THP-1 macrophages
We first examined whether M.bovis could induce caspase-1 activation and IL-1βsecretion in
THP-1 monocyte-derived macrophages at various MOIs (Fig 1A). Infection of THP-1 macro-
phages with M.bovis led to release of IL-1βinto the supernatant in a dose-dependent fashion
at MOIs ranging from 0.1 to 10, but IL-1βsecretion was not enhanced further at an MOI of
100. Meanwhile, bacterial challenge resulted in increased production of proIL-1β, a precursor
of IL-1β.M.bovis also induced caspase-1 maturation, as evidenced by increased levels of the
cleaved p20 subunit in the supernatant, which directly correlated with MOI. To investigate
whether capase-1 activation is required for IL-1βsecretion induced by M.bovis, THP-1 macro-
phages were pretreated with Z-YVAD-FMK, a cell permeable inhibitor of caspase-1; this con-
siderably reduced IL-1βsecretion (but not proIL-1βproduction) upon M.bovis infection (Fig
1B). As M.bovis can be internalized by macrophages, the contribution of intracellular bacteria
to IL-1βsecretion and caspase-1 activation was examined. THP-1 macrophages were treated
with cytochalasin D, a drug that inhibits actin polymerization and thus blocks phagocytosis of
Mycobacterium bovis Activates the NLRP7 Inflammasome
PLOS ONE | DOI:10.1371/journal.pone.0152853 April 4, 2016 4/13
M.bovis. Substantial inhibition of caspase-1 activation and IL-1βrelease were observed follow-
ing M.bovis infection (Fig 1C). Taken together, these data show that M.bovis induces IL-1β
secretion in a caspase-1-dependent manner in THP-1 macrophages.
M.bovis infection upregulates the expression of NLRP7 mRNA
The NLRP7 inflammasome is activated by a variety of microorganisms through the recogni-
tion of microbial acetylated lipopeptides. Since M.bovis expresses secreted and membrane-
associated lipoproteins, such as MPB70/80, MPB83 [16], and P27 [15]. To explore whether the
NLRP7 inflammasome is activated by M.bovis, we initially examined the time course of the
mRNA expression of NLRP7 in THP-1 macrophages. Treatment with M.bovis significantly
upregulated the expression of NLRP7 mRNA at 14 h post-infection (hpi). The levels increased
to approximately 3-fold relative to negative control, and then decreased to 1.3-fold at 50 hpi
(Fig 2A). Stimulation with M.bovis at different MOIs ranging from 0.1 to 100 revealed that
NLRP7 was upregulated in a dose-dependent manner (Fig 2B). Although there was an increase
in transcriptional level, infection with M.bovis at MOIs of 0.1 and 1 failed to produce a signifi-
cant change. In spite of upregulation of NLRP7 at the mRNA level, there is no change at the
protein level even at an MOI of 10 at 50 hpi (Fig 2C), or at an MOI of 100 at 14 hpi (Fig 2D).
The AIM2 inflammasome is activated through the recognition of DNA during M.bovis infec-
tion [19,20], and the NLRP3 inflammasome is thought to be activated after exposure to the
secreted protein, ESAT-6 [21,22]. To clarify the role of the AIM2 and NLRP3 inflammasomes
in M.bovis-infected THP-1 macrophages, we quantified their expression following infection.
There was a rapid induction of NLRP3 mRNA within 2 hpi, which then fell gradually in a
time-dependent manner, reaching control levels at 26 hpi (Fig 2E). AIM2 mRNA increased as
early as 26 hpi, and continued to increase at 50 hpi (Fig 2F). M.bovis infection failed to induce
any changes in protein levels of NLRP3 and AIM2 (Fig 2C). Infections at various MOIs
showed that M.bovis-induced upregulation of both NLRP3 and AIM2 was dose-dependent
Fig 1. M.bovis triggers caspase-1 activation and IL-1βsecretion in THP-1 macrophages. A. Cells were infected at the indicated MOIs and samples
were harvested at 14 hpi. Culture supernatant was analyzed for IL-1βand caspase-1, and cell lysates were analyzed for proIL-1β, procaspase-1, and β-actin
by immunoblotting. B. Cells were pretreated with 50 μM Z-YVAD-FMK for 1 h, and then infected with M.bovis at an MOI of 10 in the presence or absence of
Z-YVAD-FMK. Culture supernatant and lysates were analyzed by immunoblotting. C. Cells were treated with 1 μg/mL cytochalasin D to block phagocytosis
for 1 h, and then infected with M.bovis in the presence or absence of cytochalasin D. Culture supernatant and lysates were analyzed by immunoblotting.
Abbreviations: Sup, culture supernatant; Lys, cell lysates; Z-YVAD, Z-YVAD-FMK; cyto D, cytochalasin D. Data from one representative experiment of three
are presented.
doi:10.1371/journal.pone.0152853.g001
Mycobacterium bovis Activates the NLRP7 Inflammasome
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(Fig 2G and 2H). Thus, M.bovis induces upregulation of the mRNA level of NLRP7 besides
NLRP3 and AIM2.
The NLRP7 inflammasome contributes to caspase-1 activation and IL-
1βsecretion during M.bovis infection
To further investigate whether the NLRP7 inflammasome plays a role in M.bovis-induced IL-
1β, we utilized a pool of siRNAs to knock down NLRP7 in THP-1 macrophages. Compared to
non-targeting control, siRNA-mediated knockdown significantly reduced the protein levels of
NLRP7, and also attenuated caspase-1 activation and IL-1βsecretion following stimulation
with M.bovis (Fig 3A). NLRP7 promotes IL-1βsecretion via activation of the inflammasome,
Fig 2. M.bovis infection leads to upregulation of NLRP7 mRNA expression. A, E, F. THP-1 macrophages were infected with M.bovis for the indicated
times. Cell lysates were subjected to quantitative real-time PCR analysis. *0.01 <P<0.05, **P<0.01. B, G, H. Cells were infected with M.bovis at the
indicated MOI. Lysates were harvested at 14 hpi, and subjected to quantitative real-time PCR analysis. C. Cells were infected with M.bovis for the indicated
times, and supernatant and lysates were analyzed by immunoblotting. D. Cells were infected with M.bovis at the indicated MOI at 14 hpi, and supernatant
and lysates were analyzed by immunoblotting.
doi:10.1371/journal.pone.0152853.g002
Mycobacterium bovis Activates the NLRP7 Inflammasome
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which is a multiprotein complex that contains ASC and caspase-1. siRNA knockdown experi-
ments also showed that loss of ASC significantly reduced the induction of caspase-1 activity
and IL-1βsecretion following infection with M.bovis (Fig 3B).
Upon specific stimulation, NLRP7 colocalizes and interacts with ASC and caspase-1 to form
the NLRP7 inflammasome. To confirm M.bovis-induced NLRP7 inflammasome activation, we
carried out an immunofluorescence assay. In M.bovis-infected THP-1 macrophages, we
observed that NLRP7 colocalized with ASC and caspase-1 in the perinuclear area, with some
colocalization in the nucleus (Fig 3C). These effects were also observed after stimulation with
Pam3CSK4, an NLRP7 inflammasome inducer (Fig 3D). Taken together, these data suggest
that M.bovis infection induces NLRP7 inflammasome activation, which in turn promotes cas-
pase-1 activation and IL-1βsecretion.
Fig 3. The NLRP7 inflammasome facilitates caspase-1 activation and IL-1βsecretion upon M.bovis infection. A—B. THP-1 cells were
transfected with siRNA that targets NLRP7 or ASC, and then infected with M.bovis. Supernatant and cell lysates were analyzed by immunoblotting.
C—D. Cells were stimulated with M.bovis or Pam3CSK4 for 6 h. Colocalization of NLRP7 and ASC or caspase-1 was analyzed by confocal microscopy.
Magnification, ×60.
doi:10.1371/journal.pone.0152853.g003
Mycobacterium bovis Activates the NLRP7 Inflammasome
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NLRP7 inflammasome activation induces M.bovis-mediated pyroptosis
Inflammasome activation is linked to caspase-1 dependent cell death called pyroptosis [23].
We evaluated cell death by LDH release, and observed that M.bovis infection led to significant
increase of LDH release, and inhibition of caspase-1 bioactivity markedly decreased this effect
(Fig 4). To evaluate whether NLRP7 inflammasome activation relates to pyroptosis, NLRP7-
and ASC-silenced cells were stimulated with M.bovis in the presence or absence of the cas-
pase-1 inhibitor, Z-YVAD-FMK. The results indicated that NLRP7 or ASC silencing attenu-
ated LDH release, but made no difference in the presence of caspase-1 inhibitor compared to
non-targeting control following infection, suggesting that NLRP7 inflammasome activation is
involved in the cell death induced by M.bovis infection which is dependent on caspase-1 (Fig
4). Cell lysis during pyroptosis results from caspase-1-mediated pore formation in the cell
membrane and subsequent influx of extracellular fluid [23]. The cytoprotective agent glycine
inhibits pyroptosis because it nonspecifically prevents ion fluxes and suppresses swelling and
lysis [24]. Addition of glycine to the culture medium substantially decreased LDH release in M.
bovis-infected cells; this was not further enhanced by silencing of NLRP7 or ASC (Fig 4).
Taken together, these data show that NLRP7 inflammasome activation contributes to pyropto-
sis induced by M.bovis infection.
NLRP7 inflammasome activated by M.bovis promotes mRNA
expression of IL-1β, TNF-αand CCL3, but inhibits IL-18 expression
Virulent mycobacteria causes a potent inflammatory response characterized by macrophage
generation of cytokines, including TNF-αand CCL3, which contributes to granuloma forma-
tion through the recruitment of more macrophages and lymphocytes [25]. To investigate
whether the NLRP7 inflammasome plays a role in the production of TNF-αand CCL3 besides
IL-1βand IL-18, we silenced NLRP7 or ASC in THP-1 macrophages prior to infection, which
Fig 4. NLRP7 inflammasome activation promotes induction of pyroptosis in M.bovis-infected THP-1 cells. Cell death was evaluated by LDH release
in NLRP7- or ASC-silenced cells stimulated with M.bovis in the presence or absence of Z-YVAD-FMK or glycine. Abbreviations: siCon, control non-targeting
siRNA; siNLRP7, NLRP7-targeting siRNA; siASC, ASC-targeting siRNA.
doi:10.1371/journal.pone.0152853.g004
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significantly attenuated M.bovis-induced upregulation of TNF-α, CCL3 and IL-1βat the
mRNA level (Fig 5A–5C). Additionally, M.bovis infection still led to obvious increases in IL-
1β, TNF-αand CCL3 mRNAs in NLRP7- and ASC-silenced cells. However, M.bovis infection
failed to change IL-18 mRNA expression, while NLRP7- or ASC-silencing led to its upregula-
tion (Fig 5D), which may be due to various sample-collection times. These data suggest that
the NLRP7 inflammasome is involved in M.bovis-induced upregulation of the mRNA expres-
sions of TNF-α, CCL3 and IL-1βas well as downregulation of the mRNA expression of IL-18.
Discussion
Previous studies described the interaction between virulent mycobacteria and their hosts. Viru-
lent mycobacteria lead to the activation of NLRP3 inflammasome through recognition of the
ESAT-6 protein [21], However, a later study suggested that NLRP3 inflammasome activation
was dispensable for the control of pulmonary tuberculosis [26]. The AIM2 inflammasome is
activated through the recognition of M.tuberculosis DNA in infected peritoneal macrophages
[20], but Shah et al. demonstrated that M.tuberculosis inhibits AIM2 inflammasome activation
in BMDCs [27]. Here we found that virulent M.bovis also activates the NLRP7 inflammasome
in THP-1 macrophages, and contributes to induction of pyroptosis and expression of TNF-α
Fig 5. NLRP7 inflammasome activation influences the expression of TNF-α, CCL3, IL-1βand IL-18 mRNA. A—B. THP-1 macrophages were
transfected with non-targeting siRNA, or NLRP7-targeting siRNA, or ASC-targeting siRNA, and then infected with M.bovis. Lysates were subjected to
quantitative real-time PCR analysis.
doi:10.1371/journal.pone.0152853.g005
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and CCL3 due to NLRP7 inflammasome activation, although the precise mechanisms respon-
sible for NLRP7 inflammasome activation in M.bovis-infected macrophages are still unclear.
The effect of NLRP7 on IL-1βsecretion is debatable; the initial in vitro investigation by
Kinoshita et al. linking NLRP7 to IL-1βsecretion was based on inflammasome reconstitution
assays in HEK293 cells, which displayed a negative regulation. NLRP7 inhibits processing of
procaspase-1 and proIL-1βand LPS-induced IL-1βsecretion through interaction with both of
these proteins [28]. Another study by Khare et al. using stably silenced targeted gene in THP-1
cells proved that NLRP7 assembles with procaspase-1 and ASC to form the inflammasome,
and contribute to increased IL-1βsecretion in response to some heat-killed bacteria. Co-
expression of NLRP7 with ASC, procaspase-1 and proIL-1βalso led to increased level of active
IL-1βrelease compared to the level of active IL-1βin the absence of NLRP7 [12], which is
opposite to the results presented by Kinoshita et al [28]. In our study, we found that knock-
down of endogenous NLRP7 in THP-1 macrophages led to reduced IL-1βsecretion in M.
bovis-infected cells. This is consistent with the data of Khare et al.[12], showing that NLRP7
positively regulates IL-1βrelease through inflammasome activation in response to M.bovis
infection.
The mechanisms involved in inflammasome activation are complicated, with some bacteria
and viruses activating more than one type of inflammasome due to their possession of different
PAMPs. For example, Listeria monocytogenes induces activation of the NLRP3, AIM2 [29],
NLRC4 [30], and NLRP7 inflammasomes within 16 hpi [12]. M.bovis infection caused upregu-
lation of NLRP3, NLRP7 and AIM2 mRNAs at different time points, which may reflect the
temporal production of specific bacterial inflammasome stimuli. Thus, the NLRP3 inflamma-
some is activated by M.tuberculosis as early as 4 hpi [26], and the role of AIM2 inflammasome
in M.tuberculosis- and M.bovis-infected macrophages were detected at 24 hpi [19,20].
NLRP7 was upregulated at the mRNA level, but not at the protein level in THP-1 macro-
phages. This may be similar to NLRP3, which is increased at the protein level in activated
RAW 264.7 cells, primary cultured astrocytes of mice, and LPS-primed bone marrow macro-
phages [31–33], but not in LPS-treated THP-1 cells [34]. The reason for these differences may
be due to cell type or to the effect of PMA on protein expression [35].
Pyroptosis, a type of programmed cell death which is dependent on caspase-1, is an efficient
mechanism of intracellular bacterial clearance [36]. Here, we found that the NLRP7 inflamma-
some plays a role in the induction of pyroptosis in M.bovis-infected cells. Although proinflam-
matory cytokine release and pyroptosis are both induced by M.bovis infection, and require
caspase-1 activation, these two processes may occur independently, and previous studies dem-
onstrated that Legionella pneumophila,Burkholderia thailandensis and Salmonella typhimur-
ium that persistently expresses the flagellin protein were cleared through the pyroptosis
pathway and independently of cytokine release [36].
Granulomas are typical histopathological changes in tuberculosis, which represent a ‘stale-
mate’between the host and the bacteria, benefitting both parties [37]. TNF-αand chemokines
play a vital role in granuloma formation and facilitate restriction of M.tuberculosis infection
[38,39]. In this study, M.bovis-induced activation of the NLRP7 inflammasome promoted the
expression of the TNF-αand CCL3. Silencing of NLRP7 or ASC did not block the induction of
TNF-αin M.bovis-infected THP-1 cells, which may be due to incomplete silencing, and/or the
existence of alternate pathways such as NF-κB signaling [40]. For example, avirulent M.bovis
BCG is unable to activate the inflammasome in the infected macrophages [26], but it still
increases the transcription of TNF-α[41].
In conclusion, we demonstrate that M.bovis infection activates the NLRP7 inflammasome,
which in turn facilitates IL-1βsecretion, induction of pyroptosis, and upregulation of TNF-α,
Mycobacterium bovis Activates the NLRP7 Inflammasome
PLOS ONE | DOI:10.1371/journal.pone.0152853 April 4, 2016 10 / 13
CCL3 and IL-1βat the mRNA level. Our study contributes to a better understanding of innate
immune response to mycobacterial infection.
Author Contributions
Conceived and designed the experiments: YZ SZAS LY ZZ XZ DZ. Performed the experiments:
YZ SZAS. Analyzed the data: YZ. Contributed reagents/materials/analysis tools: YZ SZAS LY
ZZ XZ DZ. Wrote the paper: YZ SZAS XZ.
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