Microbial Pathogenesis 89 (2015) 177e183

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Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production Ning Liu a, Lin Wang b, Changjiang Sun c, Li Yang b, Bin Tang b, Wanchun Sun b, Qisheng Peng b, * a b c

Central Laboratory, The Second Hospital of Jilin University, Changchun 130041, China Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, 130062, China College of Veterinary Medicine, Jilin University, Changchun, 130062, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 June 2015 Received in revised form 8 September 2015 Accepted 11 October 2015 Available online 30 October 2015

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Brucella DNA Macrophage Activation NO

1. Introduction Brucella is a Gram-negative, facultative intracellular bacterial pathogen that causes abortion and infertility in animals and leads to recurrent fever and debilitating musculoskeletal, cardiac and neurological complications at the chronic stage of the infection in humans. Its virulence depends on survival and replication properties in host cells [1]. In comparison to other classical pathogens, such as Salmonella or Neisseria pathogens, Brucella is devoid of many classical structures involved in virulence including pili, fimbria, capsules, and plasmids [2]. In addition, the outer membrane fragments of Brucella containing lipoproteins, lipids, Brucella LPS (Br-LPS), and related polysaccharides are weak inducers of innate immunity [3]. Associated with this phenotype is the observation that Brucella replication in vivo and ex vivo does not depend upon TLR4, TLR3, TLR2, TLR5, or TRIF adapter [3e6]. Therefore, Brucella is able to escape recognition of the innate immunity and then survive within host cells via evading intracellular destruction.

* Corresponding author. E-mail address: [email protected] (Q. Peng). http://dx.doi.org/10.1016/j.micpath.2015.10.011 0882-4010/© 2015 Elsevier Ltd. All rights reserved.

However, the killed Brucella induces higher levels of proinflammatory cytokines than live bacteria in TLR2 and TLR4-deficient macrophages [3], suggesting that some hidden pathogenassociated molecular patterns (PAMPs) are uncovered after the bacteria have been disrupted. Furthermore, it was demonstrated that bacterial DNA extracted from heat killed Brucella abortus induces IL-12 production by dendritic cells dependent on TLR9 [7]. Moreover, Brucella DNA was identified to act as a major bacterial component to induce type I IFN [8]. Besides, it has been reported that Brucella DNA in the cytosol participates in caspase-1 activation and IL-1b secretion by inducing AIM2-inflammasome activation [9]. Collectively, these studies indicated that Brucella DNA may be involved in innate immune response to bacterial species. Our previous study showed that the silence of triggering receptor expressed on myeloid cells-2 (TREM-2) activates the macrophages, which leads to the NO production to kill the live bacterial within macrophages [10]. Considering that Brucella DNA can be sensed by TLR9 on endosomal membranes, which plays an important role in host resistance to Brucella in mice [11], in this study, we will investigate whether Brucella DNA stimulation can activate macrophages and its role in mediating bacterial replication within host cells.

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2. Materials and methods 2.1. Materials Aminoguanidine (AG) was purchased from Sigma. Apocynin and pancaspase inhibitor Z-VAD-FMK were purchased from Calbiochem. Anti-iNOS antibody was purchased from R&D. Anti-pERK, pJNK, pP38 and GAPDH antibodies were purchased from Cell Signaling. Enzyme-linked immunosorbent assay (ELISA) kits for TNFa, IL-6, IL-12p40 measurements were purchased from eBioscience. 2.2. Mice and bacteria TLR9 KO mice were gifted by Dr. Shuangxi Wang (Shangdong University, China) C57BL/6 mice were obtained from our own laboratory. Mice were used under protocols approved by the Jili University Animal Care and Use Committee. B. abortus strain 2308 was originally obtained from Ding's laboratory at China Institute of Veterinary Drug Control (Beijing, China) and was grown either in tryptic soy broth or on tryptic soy agar plates. B. abortus DVirB10 and Brucella melitensis DVirB10 were kindly provided by Zeliang Chen (Institute of Disease Control and Prevention, Academy of Military Medical Science, China). Brucella DVirB10 mutant was constructed as previously described [12]. 2.3. Extraction of genomic DNA from B. abortus B. abortus DNA (Ba-DNA) was extracted from B. abortus using the Takara genomic DNA protocol and reagents. Briefly, bacteria was resuspended in 0.5 ml of TE buffer, heat killed at 80
C for 30 min, and incubated at 37
C for 1 h with 0.5% sodium dodecyl sulfate and proteinase K (100 mg/ml). The following procedures were performed as previously Leonardo A. de Almeida et al. described [8]. 2.4. Peritoneal macrophages culture Murine peritoneal macrophages were elicited by intraperitoneal injection of 2 ml of 3% thioglycolate medium and subsequently were harvested by peritoneal lavage 2e3 days after injection. These cells (2.0  105 cells per milliliter in 24-well plates) were incubated for 3 h, and adherent cells were used as peritoneal macrophages. 2.5. Macrophage infection and survival assay Peritoneal macrophages were plated in 24-well plates in complete tissue culture media without antibiotics at a concentration of 2.0  105 cells per well and incubated overnight at 37
C with 5% CO2. The macrophages were infected with B. abortus strain 2308 in triplicate wells of a 24-well plate at a multiplicity of infection (MOI) of 100:1 by centrifuging bacteria onto macrophages at 400 g for 10 min at 4
C. Following 15 min of incubation at 37
C in an atmosphere containing 5% CO2, the cells were washed three times with aMEM to remove extracellular bacteria and incubated for additional 60 min in medium supplemented with 50 mg/ml gentamicin to kill extracellular bacteria. To monitor Brucella intracellular survival, infected cells were lysed with 0.1% Triton X-100 in phosphate-buffered saline (PBS) at certain times point and serial dilutions of lysates were rapidly plated onto tryptic soy agar plates to enumerate CFUs [13]. 2.6. Western blot Macrophages were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer. The protein content was assayed by BCA protein

assay reagent (Pierce, USA). Twenty micrograms of protein was loaded to SDSePAGE and then transferred to PVDF membrane. Membrane was incubated with a 1:1000 dilution of primary antibody, followed by a 1:2000 dilution of horseradish peroxidaseconjugated secondary antibody. Protein bands were visualized by ECL (GE Healthcare). The band densities were measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). The background was subtracted from the calculated area [14]. 2.7. Determination of NO production Supernatant of each cell sample was collected at certain times point after Brucella challenge. Nitric oxide (NO) content was measured by analysis its stable end product, nitrite, using a Griess reagent (Sigma) as previously described [10]. Data are expressed as micromoles of nitrite (mean ± SEM). 2.8. iNOS inhibition Peritoneal macrophages were pretreated with 0.36 mM aminoguanidine for 3 h before Brucella challenge [10]. The rest process is followed 2.5. “Macrophage infection and survival assay”. 2.9. Determination of ROS production Intracellular ROS was measured using the dihydroethidium fluorescence/HPLC assay with minor modifications [15]. Briefly, macrophages were incubated with 0.5 mM dihydroethidium for 30 min. After incubation, the cells were harvested and extracted with methanol. Oxyethidium (a product of dihydroethidium and superoxide anions) and ethidium (a product of dihydroethidium autoxidation) were separated and quantified by C-18 HPLC column (mobile phase: gradient of acetonitrile and 0.1% trifluoroacetic acid) coupled with a fluorescence detector. Superoxide anion production was determined by the conversion of dihydroethidium into oxyethidium. 2.10. Lactate dehydrogenase (LDH) assay The concentration of LDH released from the macrophages was measured using a commercial assay (Roche) according to the manufacture's protocol. Briefly, cell-free supernatants were obtained at different times, an aliquot of the medium (50 ml) 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. 2.11. Brucella DNA stimulation and TNFa, IL-6 and IL-12p40 measurement Peritoneal macrophages were stimulated with DNA of B. abortus for the indicated time. The supernatants were collected for analyzing the secretion of TNFa, IL-6 and IL-12p40 using enzymelinked immunosorbent assay (ELISA) kit (eBioscience) according to the manufacturer's instructions [16]. 2.12. Statistical analysis Statistical analysis was performed using SPSS10.0 software. Data are expressed as mean ± SEM. The statistical significance of differences was evaluated by using one-way ANOVA followed by the Student's t-test. P < 0.05 was considered statistically significant.

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3. Results 3.1. Macrophages activation induced by Ba-DNA is TLR9 dependent To investigate the role of Ba-DNA in macrophages activation, we examined the kinetics of activation of the three MAPKs and the NFkB pathway, which are all downstream of TLR stimulation. For these experiments, we used 20 mg/ml Ba-DNA, a concentration that has been reported to produce differences in cytokine secretion between the wild-type and TLR9-deficient macrophages [7]. The kinetics and magnitude of p38, ERK and JNK phosphorylation were markedly increased in wild type compared with TLR9-deficient macrophages (Fig. 1A). We also measured activation of the NF-kB pathway by examining the degradation of the inhibitor IkBa, which serves to

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retain NF-kB in the cytoplasm, thereby preventing transcriptional activation by NF-kB. We did not observe that IkBa was degraded in response to Ba-DNA stimulation in TLR9-deficient macrophages. In contrast, the degradation of IkBa was enhanced significantly in the wild type cells. The IkBa degradation was detected at 10 min, and reached maximal effect at 40 min after stimulation and returned to baseline by 60 min (Fig. 1B). Considering that increase of TNFa production is the hallmark of activated macrophages [17], TNFa production was examined after macrophages were stimulated with Ba-DNA. As shown in Fig. 1C, wild type cells secreted relatively higher concentrations of TNFa, whereas TLR9-deficient cells almost did not produce TNFa. In addition, we investigated whether enhanced cytokine production in wild type macrophages was specific to TNF or whether secretion

Fig. 1. Macrophage activation induced by Brucella DNA is TLR9 dependent. Peritoneal macrophages from wild type (WT) or TLR9 knock-out (TLR9/) mice were stimulated with 20 mg/ml Brucella DNA (Ba-DNA) for indicated time (min), at which time cells were lysed. Cytoplasmic extracts were analyzed using antibodies specific for pP38, pERK, pJNK (A) and IkBa (B). Data are representative of three independent experiments. Quantitative data for pERK, pJNK, pP38 and IkBa expression are shown under western blotting data, respectively. Data are expressed as mean ± SEM (n ¼ 3). The maximal expression of protein in the group was defined as 100%. (C) Peritoneal macrophages from WT or TLR9/ mice were stimulated with 20 mg/ml Ba-DNA, Ba-DNA (20 mg/ml) treated with DNase or 1 ng/ml E.coli LPS (EcLPS) for 12 h. Supernatants were collected after overnight incubation and assayed for TNFa by ELISA. Peritoneal macrophages from WT or TLR9/ mice were stimulated with 20 mg/ml Ba-DNA for 12 h. Supernatants were collected after overnight incubation and assayed for TNFa (D) or IL-12p40 (E) by ELISA.

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of other proinflammatory cytokines was also elevated. Our data indicated that both IL-6 and IL-12p40 secretion were significantly higher in wild type macrophages than in TLR9-deficient macrophages (Fig. 1D, E). Collectively, these data suggest that in the absence of TLR9, macrophages are insensitive to signals through Brucella DNA, and that, in wild-type macrophages, Brucella DNA promotes the macrophages activation. 3.2. Macrophages activated by Brucella DNA before infection suppresses Brucella intracellular replication via enhancing NO production Since Ba-DNA treatment is able to activate macrophages, next, we verified whether the activated cells inhibit Brucella intracellular growth within host cells. Peritoneal macrophages were infected with Brucella after macrophages were stimulated with or without Ba-DNA for 12 h, and Brucella intracellular growth was assessed at 24 h post infection by counting Brucella colony forming units (CFU).

Compared to resting macrophages, the numbers of Brucella in BaDNA-treated macrophages from wild type mice were significantly decreased at 24 h post infection. As expected, Ba-DNA treatment did not result in obvious variation of CFU in TLR9-deficient macrophages due to cells inactivation (Fig. 2 A). More importantly, cells were treated with Ba-DNA plus selective inhibitor of iNOS, aminoguanidine, prior to Brucella infection, bacterial replication remains unchanged compared with resting cells. However, treatment with Ba-DNA in the presence of apocynin (inhibitor of ROS production) or Z-VAD-FMK (a pancaspase inhibitor) still lead to markedly decreased CFU in macrophages (Fig. 2A). Taken these data together, it demonstrates that macrophages activation caused by Ba-DNA stimulation may inhibit bacterial replication via NO production. To further investigate whether NO production makes a crucial role in bacterial survival, peritoneal macrophages from wild type or TLR9-deficient mice were stimulated with Ba-DNA. The NO levels in culture supernatant were measured with the Greiss reagent. As

Fig. 2. Macrophages activated by Brucella DNA before infection suppresses Brucella intracellular replication. (A) Peritoneal macrophages from WT or TLR9/ mice treated with 20 mg/ml Ba-DNA 12 h prior to infection, or WT macrophages treated with Z-VAD-FMK (20 mM), apocynin (10 mM), 0.36 mM aminoguanidine (AG) 20 mg/ml Ba-DNA plus Z-VADFMK (20 mM), 20 mg/ml Ba-DNA plus apocynin (10 mM) or 20 mg/ml Ba-DNA plus 0.36 mM AG 12 h prior to infection, were infected with B. abortus for 24 h. Bacterial intracellular survival within macrophages was determined by counting the viable intracellular bacteria (CFU). Peritoneal macrophages from WT or TLR9/ mice were stimulated with mock, 20 mg/ml Ba-DNA or 20 mg/ml Ba-DNA plus AG (0.36 mM) for 12 h, at which time cells were lysed and immunoblotted with anti-iNOS antibody (C), meanwhile, supernatant were collected and filtered to determine NO production using the Greiss reagent (B). (D)Confluent monolayer of WT macrophages was stimulated with 20 mg/ml Ba-DNA for 12 h. ROS productions were assayed by DHE fluorescence. Positive control: Macrophages treated with 1 mM H2O2 for 12 h (E) LDH release assays of Brucella DNA-treated WT macrophages at 12 h. Positive control: Macrophages treated with 1 mM H2O2 for 12 h *P < 0.05 vs control. Data are expressed as mean ± SEM. NS is indicated as no significant difference.

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shown in Fig. 2B, Ba-DNA-treated wild type cells produced more NO, whereas the production of NO is decreased drastically in TLR9deficient cells or control cells. Consistent with this observation, the expression of inducible form of NOS (iNOS), which is responsible for NO production, was able to be detected in Ba-DNA-treated wild type cells (Fig. 2C). As ROS play an essential role in killing intracellular bacteria [18], we also measured the production of ROS using DHE fluorescence to confirm the role of ROS in bacterial replication. As presented in Fig. 2D, Ba-DNA stimulation did not contribute to ROS production in macrophages. In addition, we also observed that Brucella DNA treatment did not increase cell death compared with control macrophages (Fig. 2E). 3.3. Macrophages treated with Brucella DNA at early stage of infection inhibit Brucella intracellular replication Elıas Barquero-Calvo et al. reported that Brucella replication remains unaltered in the macrophages activated by E. coli LPS (EcLPS) at 24 h post infection [3]. Next, we determined whether macrophages stimulated with Ba-DNA affects Brucella intracellular growth after Brucella reside in cells. Peritoneal macrophages were stimulated with Brucella DNA after cells were infected with B. abortus for 2, 4 and 12 h. The CFU in the cells were enumerated at 24 h post infection. Interestingly, the data presented in Fig. 3A demonstrated that the bacterial replication was significantly

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inhibited at initial stage of infection such as 0, 2, and 4 h. However, The CFU sustains unchanged after cells were infected with Brucella for 12 h. To confirm whether Ba-DNA stimulation is able to activate Brucella-infected macrophages, the production of NO and TNFa in the culture supernatant were assayed at 24 h post infection. As shown in Fig. 3B, the concentration of NO in Brucella-infected macrophages is similar to macrophages, which were pretreated with Ba-DNA before infection. Ba-DNA treatment also can trigger Brucella-infected macrophages to produce considerable TNFa (Fig. 3C). These data suggest that macrophage activation was not endogenously inhibited by the intracellular bacteria. However, macrophages, which were infected with Brucella after 12 h, are not refractory to further activation and also such activation does not result in the inhibition of bacterial growth. 3.4. Macrophage activated after infection with VirB-deficient Brucella by Ba-DNA suppresses bacterial intracellular growth Considering that the replication Brucella-containing vacuole (rBCV) is generated by 12 h.p.i [19], we hypothesize that the formation of rBCV might prevent activated macrophage after infection to suppress bacterial intracellular replication. We select VirBdeficient Brucella, which is not able to form BCV in macrophages [20], as another model to test the notion. Peritoneal macrophages were infected with VirB10-deficient Brucella for 12 h, and then were

Fig. 3. Macrophages activation at initial stage of infection by Ba-DNA prevents Brucella intracellular replication. (A) Peritoneal macrophages from WT mice were infected with Brucella for 0, 2, 4 and 12 h; half of the infected macrophages were activated with 20 mg/ml Ba-DNA for 24 h and the rest of the cells treated with PBS for 24 h. The bacterial intracellular survival within macrophages was determined by counting CFU. Pre-activated group: Macrophages were treated with 20 mg/ml Ba-DNA 12 h prior to infection, and then infected with Brucella for another 24 h. The supernatant from macrophages infected with Brucella were collected and filtered to determine NO production using the Greiss reagent (B) or TNFa by ELISA kit (C).*P < 0.05 vs control. Data are expressed as mean ± SEM.

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stimulated with 20ug/ml Ba-DNA for another 12 h. The production of TNFa and NO were measured to determine whether macrophages were activated. As expected, Brucella DNA stimulation is able to activate Brucella-infected macrophages, because the secretion of TNFa and NO were significantly enhanced compared with non-treated macrophages (Fig. 4A and B). Next, we investigated whether macrophages, which were activated after infection, promotes eradication of bacteria. Peritoneal macrophages were stimulated with Brucella DNA after cells were infected with VirB10-deficient Brucella for 2, 4 and 12 h. The CFU of bacteria in cells was measured at 24 h post infection. Our data showed that activated macrophages markedly enhanced bactericidal function compared with the resting cells (Fig. 4C). More importantly, the bacterial survival was decreased significantly even macrophages were activated after 12 h post infection, which is different from wild type Brucella-infected macrophages. To address that NO production plays a crucial role in eradication of bacteria within macrophages, cells treated with Ba-DNA plus aminoguanidine before or after cells were infected with VirB10 mutant Brucella. As shown in Fig. 4D, inhibition of NO production results in unchanged CFU between the resting and Ba-DNA treated group. These data suggest that NO production induced by macrophage activation with Brucella DNA promotes bactericidal function of macrophages infected with VirB-deficient Brucella.

4. Discussion In the present study, we provide the evidences to demonstrate that Brucella DNA treatment activate macrophages in TLR9dependent manner. The activated macrophages suppress the Brucella intracellular replication via enhancing NO production. However, macrophage activation at the late stage of infection by Brucella DNA does not promote bactericidal function. In addition, we also show that Brucella DNAemediated macrophage activation inhibits VirB-deficient bacterial growth independently of infection status. Several groups have reported that Brucella DNA is involved in innate immune response to Brucella infection: Brucella DNA extracted from heat killed B. abortus induces IL-12 production by dendritic cells dependent on TLR9 [7]; Brucella DNA in the cytosol is partially required for caspase-1 activation and IL-1b secretion [9]; Brucella DNA stimulation can induce the production of type I IFN in macrophages [8]. However, the role of Brucella DNA in regulating macrophage activation remains unknown until now. This study is first time to clearly show Brucella DNA can act as bacterial agonist to activate macrophages. The activation of macrophages induced by Brucella DNA was demonstrated by the following evidences: (1) macrophages treated with Brucella DNA produces relatively high levels of proinflammatory cytokines such as TNFa, IL-6 and IL12p40 compared with TLR9-deficient macrophages; (2) three MAPKs, p38, JNK and ERK, and activation of the NF-kB pathway in

Fig. 4. Macrophage activated after infection with VirB-deficient Brucella by Ba-DNA suppresses bacterial intracellular growth. Peritoneal macrophages from WT mice were stimulated with 20 mg/ml Ba-DNA (or PBS) for 12 h, or infected with Brucella DVirB10 for 12 h, and then stimulated with 20 mg/ml Ba-DNA for another 12 h (Ba-DNA(12 hpi)). The supernatant from macrophages were collected and filtered to determine NO production using the Greiss reagent (B) or TNFa by ELISA kit (A). (C) Peritoneal macrophages from WT mice were infected with Brucella DVirB10 for 0, 2, 4 and 12 h; Half of the infected macrophages were activated with 20 mg/ml Ba-DNA for 24 h and the rest of the cells treated with PBS for 24 h. The bacterial intracellular survival within macrophages was determined by counting CFU. (D) Peritoneal macrophages from WT mice were infected with Brucella DVirB10 for 12 h, and then stimulated with PBS, 20 mg/ml Ba-DNA or 20 mg/ml Ba-DNA plus 0.36 mM AG for another 24 h. Bacterial intracellular survival within macrophages was determined by counting the viable intracellular bacteria (CFU). Pre-activated group: Macrophages were treated with 20 mg/ml Ba-DNA 12 h prior to infection, and then infected with Brucella for another 24 h *P < 0.05 vs control. Data are expressed as mean ± SEM.

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wild type macrophages are significantly activated in response to Brucella DNA stimulation; (3) importantly, NO levels is dramatically enhanced while wild type macrophages is stimulate with Brucella DNA. As activated macrophages are the primary source for B. abortus elimination in the infected host [21,22], these results suggest that Brucella DNA can be used as an immune cell activator to control Brucella infection. Another important finding of this study is that macrophages activation by Brucella DNA stimulation suppresses Brucella intracellular replication via enhancing NO production in host cells. Richard Copin et al. observed that TLR9 deficiency moderately affected the resistance of mice to infection in the mice spleen [11]. However, the underlying mechanism remains elusive. Macrophages treated with Brucella DNA did not result in obvious change ROS or LDH compared with resting macrophages. Moreover, suppression of ROS production or apoptosis with relative inhibitor did not interfere with bacteria intracellular growth. However, Brucella DNA treatment promotes NO production and induces iNOS protein expression, whereas we did not observe this phenotype in the TLR9-deficient macrophages. In addition, inhibition of bacterial replication caused by Brucella DNA was abolished if macrophages were treated with aminoguanidine plus Brucella DNA. Taken together, macrophages activated by Brucella DNA before infection suppresses Brucella intracellular replication via enhancing NO production in a TLR9-dependent manner. Brucella after phagocytic internalization resides within a membrane bound compartment, the Brucella-containing vacuole (BCV), which traffics along the endocytic pathway and interacts with the transitional ER from the beginning of 4 h post infection. The ERderived BCVs, which are replication-permissive organelles, was formed by 12 h post infection [20,23]. Based on these studies, we propose that activated macrophages fail to inhibit bacterial replication after the ER-derived BCVs was generated, which may explain why macrophages activation at early stage of infection inhibits Brucella intracellular replication but not inhibits at 12 h post infection. However, BCV containing VirB-deficient Brucella does not fuse with the ER but fuse with lysosomes, and ultimately is not able to generate the ER-derived BCV, which leads to elimination of bacteria from host cells [20,23]. Therefore, activated macrophages by Brucella DNA stimulation promotes bactericidal function of macrophages whatever at the early or late stage VirB mutant Brucella infection. In the future, we will investigate how the formed BCV evades the attack of activated macrophages to benefit bacterial survival within host cells, which helps us to further understand the mechanism of Brucella intracellular growth. In conclusion, the results demonstrate that macrophage activation induced by Brucella DNA inhibits bacteria growth within host cells via enhancing production of NO in TLR9-dependent manner. Brucella DNA may act as an immune cell activator to control Brucella infection. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was funded by National Natural Science Foundation of China (31372409) to QishengPeng. This work was also supported by Natural Science Foundation of China (81472030, 21175055), Jilin Province Science and Technology Department (20110739,

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20150204001YY) and Jilin University Bethune Project B (2012210) to Ning Liu.

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