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Lactobacillus reuteri protects mice against Salmonella typhimurium challenge by activating macrophages to produce nitric oxide
Microbial Pathogenesis 137 (2019) 103754
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Lactobacillus reuteri protects mice against Salmonella typhimurium challenge by activating macrophages to produce nitric oxide
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Pingping Jiang1, Wentao Yang1, Yubei Jin, Haibin Huang, Chunwei Shi, Yanlong Jiang, Jianzhong Wang, Yuanhuan Kang, Chunfeng Wang∗∗, Guilian Yang∗ College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Key Laboratory of Animal Production and Product Quality Safety of Ministry of Education, Jilin Agricultural University, Changchun, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lactobacillus reuteri Salmonella typhimurium Macrophages Phagocytosis Immunoprotection
Lactobacillus reuteri, a typical intestinal symbiotic bacterium, plays an important role in maintaining intestinal flora stability and host health. However, the effect of Lactobacillus reuteri on peritoneal macrophages has not been thoroughly studied. Our study indicated that Lactobacillus reuteri could activate macrophages and that macrophages treated with Lactobacillus reuteri have an enhanced ability to phagocytose and to kill intracellular Salmonella typhimurium. Lactobacillus reuteri may reduce the inflammatory response caused by Salmonella typhimurium by regulating NO, thus effectively protecting mice against Salmonella typhimurium invasion and dissemination to the liver and spleen. Taken together, these data demonstrated the protective effect of Lactobacillus reuteri on macrophages and mice challenged with Salmonella typhimurium through in vitro and in vivo experiments.
1. Introduction Salmonella typhimurium is a gram-negative pathogen that can infect various hosts. It can elude the host natural immune system, replicate in the host and cause enteritis, typhoid, septicemia and localized purulent infections, which pose a serious threat to animal production [1]. Pathogens such as S. typhimurium pass through the physical barriers of the body, and the innate immune system responds quickly, promoting the secretion of cytokines, activating macrophages, and inducing their bactericidal activity [2,3]. As an important component of innate immunity and adaptive immunity, macrophages can phagocytose and eliminate most pathogens within a short time after pathogens invade. In addition, macrophages prevent the invasion of pathogens through the induction of inflammation and cell toxicity via the production of effectors such as nitric oxide (NO) and reactive oxygen species (ROS), as well as the secretion of proinflammatory cytokines [4,5]. NO is an important internal and intercellular regulatory molecule and is considered the second barrier of innate defense mechanisms. Macrophages induce nitric oxide synthase (iNOS) production in response to inflammation, leading to an increase in NO after infection [6].
NO plays various roles in the innate immune system, such as killing virus-infected cells, tumor cells and parasitic pathogens [7]. The antiviral effect of NO in some viral infections has been well documented [8]. As a member of the intestinal flora, Lactobacillus plays an important role in enhancing immunity, maintaining intestinal microbial balance and preventing gastrointestinal infection [9]. As a typical intestinal symbiotic bacterium, Lactobacillus reuteri is one of the most abundant species in the intestinal tract and can be detected in many animal subgroups. Probiotics secrete microbe-associated molecular patterns (MAMPs) that are secreted into the environment or on the cell surface and, after interacting with pattern recognition receptors (PRRs), induce a signaling cascade [10]. Studies have shown that L. reuteri competitively inhibits the colonization of pathogenic bacteria in the host intestine by adhering to the surface of host intestinal epithelial cells and strengthening the host immunoregulation response to effectively control Salmonella infection [11]. However, to understand the preventive effect of L. reuteri and the underlying mechanisms on viral infections, many studies must be performed. Our study used L. reuteri and heat-inactivated L. reuteri to
∗ Corresponding author. College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University, 2888 Xincheng Street, Changchun, 130118, China. ∗∗ Corresponding author. College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University, 2888 Xincheng Street, Changchun, 130118, China. E-mail addresses: [email protected] (C. Wang), [email protected] (G. Yang). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.micpath.2019.103754 Received 19 June 2019; Received in revised form 12 September 2019; Accepted 16 September 2019 Available online 17 September 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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2.5. Cytotoxicity of Lactobacillus reuteri
intervene in C57BL/6 mice and mouse peritoneal macrophages infected with S. typhimurium for the purpose of studying the early interaction in immune system macrophages, pathogens and potential protective probiotics.
To evaluate the cytotoxicity of L. reuteri and heat-inactivated L. reuteri, we examined cell viability using the MTS assay as previously described in Ref. [14]. Peritoneal macrophages (2 × 104 cells per well) were transferred to a 96-well plate and treated with saline and different concentrations of L. reuteri and inactivated L. reuteri (105, 106 and 107 CFU) for 1 h. Subsequently, the above materials were washed with PBS, and the cells were incubated for an additional 24 h at 37 °C. MTS was added to the 96-well plate for further cultivation of 4 h, and the absorbance of each well at 490 nm was measured using a microplate reader. The percentage of cell viability was calculated using the following formula: cell viability (%) = (ODsample – ODblank)/(ODcontrol- ODblank) × 100.
2. Materials and methods 2.1. Reagents Dulbecco's modified Eagle’ medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, phosphate-buffered saline (PBS) and other miscellaneous cell culture reagents were purchased from HyClone Laboratories (Logan, UT, USA). Fluorescent microspheres were obtained from Molecular Probes (Eugene, Oregon, USA). MTS was obtained from Promega (Madison, Wisconsin, USA). A nitrite assay kit was purchased from Beyotime Biochemical Co. (Haimen, China). Antimouse F4/80 (clone BM8) antibodies conjugated to PE were obtained from eBioscience (CA, USA), and anti-mouse CD80 (clone 16-10A1) antibodies conjugated to FITC, anti-mouse CD86 (clone GL1) antibodies conjugated to PE, anti-mouse CD40 (clone 3/23) antibodies conjugated to FITC, and anti-mouse CD11b (clone M1/70) antibodies conjugated to APC from BD Pharmingen (Erembodegem, Belgium) were also procured for flow cytometric analysis. All 96-well, 12-well and 24-well plates were obtained from Corning (USA).
2.6. Flow cytometry Peritoneal macrophages were obtained from the murine abdominal cavity and resuspended to a single cell suspension to assess macrophage purification as previously described [15]. After 24 h of cultivation, peritoneal macrophages were stained with fluorochrome-conjugated antibodies, anti-mouse F4/80-PE and anti-mouse CD11b-APC. Subsequently, the fluorescence in the macrophages was examined by fluorescence-activated cell sorting (FACS) analysis with a BD LSR-FORTESA (BD Biosciences), and the data were analyzed using FlowJo software, version 7.6.0. In a second experiment, peritoneal macrophages were treated with saline, L. reuteri and inactivated L. reuteri. The cell suspensions were stained with fluorescein isothiocyanate anti-mouse CD86-PE, antimouse CD80-FITC, and anti-mouse CD40-FITC antibodies to analyze the activation effects of L. reuteri on macrophages. The detection method and data analysis were the same as those in the above experiment.
2.2. Cells Peritoneal macrophages were obtained from the peritoneal cavity of 6-week-old C57BL/6 mice [12]. After one day, mice were injected with Brewer's thioglycollate broth through intraperitoneal injection. DMEM culture containing 3% FBS was injected into the peritoneal cavity of mice, and the peritoneal cells were obtained along with the medium at 84–120 h, as previously described in Ref. [13]. The obtained peritoneal macrophages were cultivated in DMEM containing fetal bovine serum and penicillin-streptomycin at 37 °C in humidified conditions with 5% CO2, and adherent cells were removed after 2 h.
2.7. Peritoneal macrophages killing intracellular bacteria Extraction of mouse peritoneal macrophages was carried out as previously described [16]. The obtained murine peritoneal macrophages were cultured for 2 h, and nonadherent and dead cells were removed. The macrophages were then plated in a 12-well plate at 2 × 105 cells per well (with antibiotic-free medium) and treated with saline, L. reuteri or inactivated L. reuteri for 1 h (2 × 106 CFU per well). Subsequently, Salmonella was added to the 12-well plate (2 × 106 CFU per well) and incubated at 37 °C for 1 h. The culture medium was discarded, and the plates were washed with sterile PBS. Then, gentamicincontaining DMEM was added to the plate to kill extracellular bacteria. After 1 h of incubation, the gentamicin-containing medium was replaced by antibiotic-free medium, which was cultured for another 2 and 4 h. Macrophages were lysed with 1% Triton X-100 to release intracellular bacteria, and serial dilutions were plated on SalmonellaShigella agar plates. The CFU were enumerated by counting black-centered colonies after incubating the plates for 16–18 h at 37 °C.
2.3. Experimental animal ethical status Six-to eight-week-old female C57BL/6 mice (Beijing HFK Bioscience Co., Ltd., China) were fed a standard mouse chow diet and acclimatized for 7 days prior to the commencement of feeding experiments. All animal experiments were approved by the Animal Care and Ethics Committees of the Jilin Agriculture University, China, and performed according to the Jilin Agriculture University guidelines for Laboratory Animals Care and Usage. The animal facility of the Jilin Agricultural University is fully accredited by the National Association of Laboratory Animal Care. 2.4. Bacterial strain
2.8. Fluorescent microspheres phagocytosis assay L. reuteri ATCC 55730 was obtained from the Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University. L. reuteri was transferred to de Man Rogosa Sharpe (MRS) broth and incubated at 37 °C overnight. Subsequently, bacteria were centrifuged, washed and resuspended in sterile saline, and 1 × 107 CFU of L. reuteri were injected per mouse via the intraperitoneal route. Heatinactivated L. reuteri was boiled for 10 min before use. Salmonella typhimurium ATCC 14028 was obtained from the Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University; it was grown at 37 °C with shaking in Luria Bertani (LB) until the mid-exponential phase (absorbance at 660 nm, 1.0) and then diluted with sterilized saline to a concentration appropriate for each experiment.
Peritoneal macrophages obtained from the peritoneal cavity of mice were transferred to 24-well plates at 2 × 105 cells per well. After 2 h of incubation, nonadherent and dead cells were removed, and peritoneal macrophages were treated with saline, L. reuteri or inactivated L. reuteri for 1 h. Fluorescence microspheres were added to the plates at 2 × 106 per well, and then the plates were cultured for 45 and 90 min. Subsequently, the cells were stained with DAPI in the dark for 15 min and observed under a fluorescence microscope. 2.9. Estimation of NO in the cell culture supernatant Peritoneal macrophages were transferred to a 12-well plate 2
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(2 × 105 cells/well) and treated with saline, L. reuteri or inactivated L. reuteri for 1 h. Subsequently, S. typhimurium (2 × 106 CFU/well) was added to the plate, and the bacteria were removed after 1 h of cultivation and incubated for 2, 4 and 6 h. To estimate the effects of L. reuteri and inactivated L. reuteri on the NO production of peritoneal macrophages, a Nitrate/Nitrite Assay Kit was used to detect nitric oxide levels in the cell supernatant.
These results indicated that different concentrations of L. reuteri and inactivated L. reuteri had no negative effect on cell activity. The multiplicity of infection in subsequent experiments was set at 10 according to this result. 3.2. L. reuteri-induced macrophage activation To detect the purity of macrophages extracted from the abdominal cavity of mice, after removing nonadherent cells and culturing for a period of time, cell surface staining was performed using directly conjugated antibodies: anti-mouse F4/80 and CD11b for purity of peritoneal macrophages and CD40, CD80 and CD86 for L. reuteri-activated macrophages. Flow cytometry results revealed that the peritoneal macrophages we obtained were highly efficient in differentiation (Fig. 2A), and the peritoneal macrophages treated with L. reuteri showed higher expression of CD40 (***P < 0.001), CD80 (***P < 0.001) and CD86 (**P < 0.01) than peritoneal macrophages treated with saline (Fig. 2B and C). However, inactivated L. reuteri enhanced the expression of CD40 (**P < 0.01) and CD86 (*P < 0.05) in macrophages compared with that in the saline group macrophages, which indicated that L. reuteri activated macrophages and that its effect was better than that of inactivated L. reuteri.
2.10. Immunization and infection 24 female C57BL/6 mice were randomly divided into 3 groups, each group consisted of 8 mice, and the immunization procedures were as follows. Two groups of mice were immunized by intraperitoneal injection with L. reuteri or inactivated L. reuteri (1 × 107 CFU) every other day from the first day and then infected with a lethal dose of S. typhimurium (1 × 104 CFU) by intraperitoneal injection on the second day. The procedure of the third group was the same as above; however, mice were injected with the same volume of saline as that for the L. reuteri injection every other day and then infected with S. typhimurium the next day. Weight loss was recorded from the first day, and the survival rate was calculated. 2.11. CFU analysis
3.3. L. reuteri reduces intracellular bacterial survival Liver and spleen tissues were collected 3 days after the mice were challenged with S. typhimurium, and liver and spleen tissues were weighed and homogenized in sterile PBS (g/ml). Appropriate dilutions were plated on Salmonella-Shigella agar plates. The CFU were calculated by counting black-centered colonies after incubating the plates for 16–18 h at 37 °C.
3. Results
Peritoneal macrophages were treated with saline, L. reuteri or inactivated L. reuteri for 1 h, then fluorescence microspheres were added. After culturing for another 45 and 90 min, intracellular fluorescence intensity was observed with a fluorescence microscope (Fig. 3A). The results showed that the ratio of fluorescence microspheres to cells in the L. reuteri and inactivated L. reuteri groups was significantly higher than that in the saline group at 45 min (Fig. 3B). Differences were also observed between the L. reuteri group and the control group after 90 min of treatment; however, there was no difference in the ratio of fluorescence microspheres to cells between the inactivated L. reuteri group and the control group (Fig. 3C), perhaps because the phagocytosis ability increased in the control group over time, while the phagocytosis ability remained unchanged in the L. reuteri and inactivated L. reuteri groups. The results indicated that both L. reuteri and inactivated L. reuteri could enhance peritoneal macrophage phagocytosis, and L. reuteri showed better phagocytosis enhancement activity than inactivated L. reuteri.
3.1. Cytotoxicity of Lactobacillus reuteri
3.4. L. reuteri improved macrophage phagocytosis
To explore the toxic effects of L. reuteri on macrophages, we transferred peritoneal macrophages into 96-well plates with 104 cells per well. Macrophages were infected with different concentrations of L. reuteri and inactivated L. reuteri (105, 106, or 107 CFU per well), and cell viability was detected by MTS assay after infection. The results showed that there was no difference in cell viability among the 105, 106, and 107 CFU of L. reuteri treatment groups and the control group (Fig. 1A). Similarly, the inactivated L. reuteri showed the same results (Fig. 1B).
Peritoneal macrophages isolated from mice were cultured in the presence of L. reuteri, inactivated L. reuteri or saline for 1 h, and the cells were infected with S. typhimurium for 1 h, followed by 2 and 4 h of incubation. Cells were lysed, and the intracellular bacteria were counted. The results showed that there was no significant difference in the number of intracellular bacteria between the saline group and the L. reuteri- or the inactivated L. reuteri-treated group at 2 h. However, the number of intracellular bacteria in the L. reuteri or inactivated L. reuteri
2.12. Statistical analysis Statistical analysis results are expressed as the mean values and standard deviations (SDs) of triplicate experiments. The data were visualized using GraphPad Prism 5 software. The statistical significance was analyzed using an unpaired two-tailed t-test and a one-way ANOVA test, and P-values of less than 0.05 were considered statistically significant.
Fig. 1. Cytotoxicity of Lactobacillus reuteri on peritoneal macrophages. Peritoneal macrophages were transferred to a 96-well plate and treated with saline and different concentrations of L. reuteri and heat-inactivated L. reuteri (105, 106 or 107 CFU) for 1 h. Cells were incubated for another 24 h at 37 °C. Cell viability was detected by MTS assay. (A) Effect of L. reuteri on cell viability. (B) Effect of inactivated L. reuteri on cell viability. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, **P < 0.01).
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Fig. 2. Differentiation rate of peritoneal macrophages and effects of L. reuteri on macrophage activation. Mice were sacrificed, and peritoneal macrophages were obtained with peritoneal exudate. (A) Differentiation rate of peritoneal macrophages. The peritoneal macrophage differentiation rate was analyzed via the markers F4/80 and CD11b and quantified by flow cytometry. (B) Effects of L. reuteri on macrophage activation. Peritoneal macrophages were treated with saline, L. reuteri or inactivated L. reuteri for 1 h, and representative flow cytometric analyses of activated macrophages cells with the markers CD40, CD80 and CD86 were performed. (C) The histogram values are the percentages of activated macrophages cells with the markers CD40, CD80, and CD86. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, **P < 0.01, ***P < 0.001).
inactivated L. reuteri for 1 h and then infected with S. typhimurium (1 × 106 CFU/well) for 1 h, and the cells were cultured for 2, 4 and 6 h. As shown in Fig. 4, nitric oxide content in the S. typhimurium group, L. reuteri plus S. typhimurium group and inactivated L. reuteri plus S. typhimurium group was significantly higher than that in the saline group (***P < 0.001), and furthermore, that in the L. reuteri plus S. typhimurium and inactivated L. reuteri plus S. typhimurium groups was significantly higher than that in the S. typhimurium group (**P < 0.01, *P ＜0.05). The nitric oxide levels of the above three groups decreased
group decreased at 4 h compared with that in the saline group (Fig. 3B). The results indicated that L. reuteri and inactivated L. reuteri enhance the ability of peritoneal macrophages to kill intracellular bacteria. 3.5. Effects of L. reuteri on NO production A nitrate/nitrite kit was used to determine the effect of L. reuteri and inactivated L. reuteri on macrophage nitric oxide production. Peritoneal macrophages (1 × 105 cells/well) were treated with L. reuteri or 4
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(caption on next page)
The above results indicated that L. reuteri and inactivated L. reuteri could increase the production of nitric oxide in macrophages infected with S. typhimurium, thus affecting the antimicrobial ability of macrophages. However, when cells were treated with L. reuteri or inactivated
with time. In contrast, the nitric oxide content in the L. reuteri and inactivated L. reuteri groups showed no difference from that in the saline group in the first 4 h but increased at 6 h, while the nitric oxide content in the saline group remained unchanged. 5
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Fig. 3. L. reuteri and inactivated L. reuteri can improve the phagocytosis and sterilization abilities of peritoneal macrophages. (A) L. reuteri and inactivated L. reuteri improve peritoneal macrophage phagocytosis. Peritoneal macrophages were pretreated with saline, L. reuteri or inactivated L. reuteri for 1 h. Fluorescent microspheres were added to the cells and cultured for 45 or 90 min, and macrophage phagocytosis of the microspheres was observed under a fluorescence microscope. (B) Higher fluorescence of microspheres per cell was observed in the L. reuteri group (n = 20) and inactivated L. reuteri group (n = 22) than in the control group (n = 25) after 45 min of treatment (***P < 0.001). (C) After 90 min of treatment, differences were also observed between the L. reuteri (n = 24) and control groups (n = 18), and no differences were detected between the inactivated L. reuteri group (n = 24) and the control group (**P < 0.01). (D) Intracellular bacteria enumeration in macrophages. Peritoneal macrophages treated as described above were infected with S. typhimurium (1 × 105 CFU) for 1 h, culture medium containing gentamicin was used to kill extracellular bacteria, and intracellular bacteria were counted at the indicated times. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05).
saline group (***P < 0.001), while there was no difference between the L. reuteri and inactivated L. reuteri groups (Fig. 5C). Consistently, spleen S. typhimurium CFU in the L. reuteri and inactivated L. reuteri groups were also significantly lower than those in the saline group (***P < 0.001). However, no difference was observed in S. typhimurium CFU between the L. reuteri group and inactivated L. reuteri group (Fig. 5D). Moreover, there was no difference in the spleen S. typhimurium CFU between the L. reuteri group and the inactivated L. reuteri group. The results indicated that L. reuteri and inactivated L. reuteri inhibited the dissemination of S. typhimurium from the abdominal cavity to the spleen and liver. 4. Discussion Fig. 4. Effects of L. reuteri and inactivated L. reuteri on NO production of macrophages infected with S. typhimurium. Murine peritoneal macrophages (1 × 105 cells/well) were treated with saline, L. reuteri or inactivated L. reuteri (1 × 106 CFU/well) for 1 h. Then, S. typhimurium (1 × 106 CFU/well) was added to peritoneal macrophages and incubated for 1 h. After an additional 2 or 4 h of incubation, the cell culture supernatant was collected, and the NO content in the supernatant was determined. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, **P < 0.01, ***P < 0.001).
Lactobacillus, as a probiotic, interacts with intestinal microbial communities and hosts and has a positive impact on individual health when ingested in sufficient quantities [17]. Lactobacillus reuteri is a widely studied probiotic naturally existing in the digestive tract of most vertebrates and mammals [18]. Lactobacillus reuteri can control bodily endotoxin levels, protect the liver and improve liver detoxification, indicating that Lactobacillus reuteri plays the role of immune regulation in the abdominal cavity, including stimulating peritoneal macrophage activation and inducing antibodies and interferon production [19,20]. Nonactivated macrophages express only a small amount of MHCII and do not express costimulatory molecules. However, macrophages are induced to express MHCII and costimulatory molecules, such as CD80 and CD86, and release inflammatory mediators, promoting the removal of alien components in cells after activation [21]. In this study, we demonstrated the protective effect of L. reuteri on S. typhimurium infection. After treatment with L. reuteri and inactivated L. reuteri, the expression of CD40, CD80 and CD86 on peritoneal macrophages was increased (Fig. 2B), indicating that both L. reuteri and inactivated L. reuteri treatment activate macrophages. Activated macrophages have the functions of releasing inflammatory mediators, presenting antigens and initiating the adaptive immune response and are an important line of defense for the body against foreign substances [22]. Macrophages are important for controlling and removing Salmonella from host cells [23]. When the pathogen invades, some highly conserved pathogen-associated molecular patterns (PAMPs) on pathogens are recognized by pattern recognition receptors (PRRs) on the surface of macrophages, which then absorb into the cell to form phagosomes that are further fused with intracellular lysosomes to form phagocytic lysosomes and digest and remove pathogens through oxygen-dependent and oxygen-independent pathways. Our results suggested that L. reuteri and inactivated L. reuteri treatment enhance macrophage phagocytic activity to kill intracellular pathogens (Fig. 3A and B). Previous studies have also confirmed that Lactobacillus can activate macrophages, enhance phagocytosis ability and inhibit intracellular pathogen survival, thus playing a role in resisting pathogens [24,25]. Three groups of mice were immunized with L. reuteri, inactivated L. reuteri or the same volume of saline by intraperitoneal injection. The mice were infected with S. typhimurium through intraperitoneal injection, and their weight and survival rate were continuously recorded. Compared with the saline group, the L. reuteri and inactivated L. reuteri groups exhibited a significantly improved survival rate and prolonged
L. reuteri alone, the production of nitric oxide in cells was not affected in a short time period, and the production of nitric oxide was increased in a long time period. 3.6. Effects of L. reuteri on weight loss and mortality of infected mice Three groups of mice were treated with saline, L. reuteri or inactivated L. reuteri (106 CFU) by intraperitoneal injection every other day. On the day after the initial immunization, mice were infected with S. typhimurium (104 CFU) by intraperitoneal injection, and the body weight and mortality were recorded for 25 consecutive days. The mice in all three groups exhibited weight loss after S. typhimurium challenge. The L. reuteri group showed slight weight loss and recovered after 5 days (Fig. 5A). Not surprisingly, the saline group succumbed to the lethal challenge of the S. typhimurium challenge within 5 days. However, only one mouse died in the inactivated L. reuteri group on day 5, and all mice in the inactivated L. reuteri group died by day 11. Interestingly, mice in the L. reuteri group began to die after all the mice in the saline group had died, and one of them survived until the end of observation (Fig. 5B). These results effectively demonstrate the potential of L. reuteri and inactivated L. reuteri in protecting mice against S. typhimurium infection, and the protective effect of L. reuteri was superior to that of inactivated L. reuteri. 3.7. Salmonella typhimurium CFU in the liver and spleen Mice were sacrificed 3 days after S. typhimurium challenge. The liver and spleen of each mouse were weighed and homogenized with PBS at a ratio of g of tissue/ml of PBS. Tissue homogenate was plated on Salmonella-Shigella agar plates after appropriate dilution, and bacterial CFU were counted after overnight culture. An approximately ten-fold decrease was observed in the liver S. typhimurium CFU of the L. reuteri group and the inactivated L. reuteri group compared with those of the 6
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Fig. 5. L. reuteri pretreatment protects mice against salmonella infection and reduces the dissemination of salmonella from the abdomen to the liver and spleen. Three groups of mice were pretreated with L. reuteri, inactivated L. reuteri or saline by intraperitoneal injection and subsequently challenged with S. typhimurium. (A) Weight loss was continuously recorded for 25 days. *Statistically significant difference relative to the inactivated L. reuteri group (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Survival rates were continuously recorded for 25 days. *Statistically significant difference relative to the saline group (**P < 0.01). (C, D) Mice were sacrificed at a designated time after challenge with S. typhimurium, the liver and spleen were collected, and the bacterial CFU of per gram of liver and spleen was counted. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, ***P < 0.001).
increased with the prolongment of infection. L. reuteri is an indigenous flora constituent in mammals that can form biological barriers in the intestinal tract and other mucosal sites to resist the invasion of pathogenic bacteria and enhance body immunity. In addition, studies have shown that L. reuteri can reduce the colonization of pathogenic bacteria in organs [34]. In this study, similar results showed that L. reuteri could reduce the colonization of S. typhimurium in the mouse liver and spleen. Macrophages induce the production of nitric oxide synthase during the inflammatory reaction, leading to an increase in NO after infection [35]. NO and ROS are considered the second barrier of the innate defense mechanism. NO plays a variety of roles in the innate immune system, such as killing virus-infected cells, tumor cells and parasitic pathogens. In some viral infections, the antiviral effect of NO has been well demonstrated [36]. However, excessive NO can damage DNA, proteins and lipids in cells and tissues. Therefore, the ultimate effect of NO should be considered as a balance between antiviral and cytotoxic effects [37,38]. In short, our results suggested that L. reuteri may play an antibacterial role by enhancing the production of nitric oxide in macrophages (Fig. 4). Nitric oxide, as an reactive nitrogen intermediate, is closely related to reactive oxygen species and other cytokines, and we may conduct further studies in this regard. In conclusion, our experiments showed that L. reuteri and inactivated L. reuteri can activate macrophages, enhance their abilities to phagocytose and to kill intracellular bacteria, and increase the secretion of nitric oxide in macrophages infected with S. typhimurium. In vivo experiments confirmed that L. reuteri and inactivated L. reuteri have
survival time in mice (Fig. 5A and B). However, the protective effect of L. reuteri on mice and peritoneal macrophages was superior to that of inactivated L. reuteri, which may be due to metabolites with antibacterial activity secreted by L. reuteri, such as reuterin and histamine [26,27]. Studies have shown that L. reuteri ameliorates viral infection by regulating the microbial community and secreting metabolites with antiviral components [28]. Reuterin is a nonprotein-based broad-spectrum antimicrobial substance that can widely inhibit the growth of gram-positive bacteria, gram-negative bacteria, yeast, fungi and pathogens [29]. Previous studies have confirmed that the culture supernatant of Lactobacillus upregulated the production of macrophage NO and partially upregulated several inflammatory cytokines in macrophages [30]. In addition, mice in the inactivated L. reuteri group had a longer survival time than those in the control group, although its protective effect on mice was less than that of live L. reuteri, which indicated that the structure of L. reuteri or proteins such as peptidoglycan may have a protective effect. Recent studies have found that L. reuteri stimulates immunomodulatory responses through macrophages and reduces the secretion of proinflammatory factors [31,32]. The liver is an important organ of the body, and its metabolism is related to the gastrointestinal tract and even the whole biological process. It is also an important target of many drugs, oxidation and pathogen invasion [33]. Liver injury will seriously affect the metabolism, detoxification, immune response and antibacterial ability of the body. In our study, the liver and spleen of the mice infected with S. typhimurium were affected, and the number of S. typhimurium in tissues 7
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protective effects on mice infected with S. typhimurium and can reduce the bacterial load in the spleen and liver of mice. In addition, in the above experiments, the protective effect of L. reuteri was superior to that of inactivated L. reuteri, which may be related to bacteriocins, such as reuterin, and other substances produced by L. reuteri.
[13]
[14]
Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
[15]
[16]
Author contributions
[17]
Wentao Yang and Chunfeng Wang conceived and supervised the study; Pingping Jiang and Wentao Yang designed experiments; Pingping Jiang, Wentao Yang, Haibin Huang and Chunwei Shi performed the experiments; Pingping Jiang, Yubei Jin, Yanlong Jiang, Jianzhong Wang, Yuanhuan Kang, Guilian Yang analyzed the data. Pingping Jiang, Wentao Yang and Chunfeng Wang Wrote the paper. All authors reviewed the results and approved the final version of the manuscript.
[18]
[19]
[20]
Funding This work was supported by the National Key Research and Development Program of China (2017YFD0501000, 2017YFD0500400), National Natural Science Foundation of China (31672528), Science and Technology Development Program of Jilin Province (2017YFD0501200, 20170204034NY, 20180520037JH), the Key Funds for Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture (ZJK201808).
[21] [22]
[23]
[24]
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Lactobacillus reuteri protects mice against Salmonella typhimurium challenge by activating macrophages to produce nitric oxide
T
Pingping Jiang1, Wentao Yang1, Yubei Jin, Haibin Huang, Chunwei Shi, Yanlong Jiang, Jianzhong Wang, Yuanhuan Kang, Chunfeng Wang∗∗, Guilian Yang∗ College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Key Laboratory of Animal Production and Product Quality Safety of Ministry of Education, Jilin Agricultural University, Changchun, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lactobacillus reuteri Salmonella typhimurium Macrophages Phagocytosis Immunoprotection
Lactobacillus reuteri, a typical intestinal symbiotic bacterium, plays an important role in maintaining intestinal flora stability and host health. However, the effect of Lactobacillus reuteri on peritoneal macrophages has not been thoroughly studied. Our study indicated that Lactobacillus reuteri could activate macrophages and that macrophages treated with Lactobacillus reuteri have an enhanced ability to phagocytose and to kill intracellular Salmonella typhimurium. Lactobacillus reuteri may reduce the inflammatory response caused by Salmonella typhimurium by regulating NO, thus effectively protecting mice against Salmonella typhimurium invasion and dissemination to the liver and spleen. Taken together, these data demonstrated the protective effect of Lactobacillus reuteri on macrophages and mice challenged with Salmonella typhimurium through in vitro and in vivo experiments.
1. Introduction Salmonella typhimurium is a gram-negative pathogen that can infect various hosts. It can elude the host natural immune system, replicate in the host and cause enteritis, typhoid, septicemia and localized purulent infections, which pose a serious threat to animal production [1]. Pathogens such as S. typhimurium pass through the physical barriers of the body, and the innate immune system responds quickly, promoting the secretion of cytokines, activating macrophages, and inducing their bactericidal activity [2,3]. As an important component of innate immunity and adaptive immunity, macrophages can phagocytose and eliminate most pathogens within a short time after pathogens invade. In addition, macrophages prevent the invasion of pathogens through the induction of inflammation and cell toxicity via the production of effectors such as nitric oxide (NO) and reactive oxygen species (ROS), as well as the secretion of proinflammatory cytokines [4,5]. NO is an important internal and intercellular regulatory molecule and is considered the second barrier of innate defense mechanisms. Macrophages induce nitric oxide synthase (iNOS) production in response to inflammation, leading to an increase in NO after infection [6].
NO plays various roles in the innate immune system, such as killing virus-infected cells, tumor cells and parasitic pathogens [7]. The antiviral effect of NO in some viral infections has been well documented [8]. As a member of the intestinal flora, Lactobacillus plays an important role in enhancing immunity, maintaining intestinal microbial balance and preventing gastrointestinal infection [9]. As a typical intestinal symbiotic bacterium, Lactobacillus reuteri is one of the most abundant species in the intestinal tract and can be detected in many animal subgroups. Probiotics secrete microbe-associated molecular patterns (MAMPs) that are secreted into the environment or on the cell surface and, after interacting with pattern recognition receptors (PRRs), induce a signaling cascade [10]. Studies have shown that L. reuteri competitively inhibits the colonization of pathogenic bacteria in the host intestine by adhering to the surface of host intestinal epithelial cells and strengthening the host immunoregulation response to effectively control Salmonella infection [11]. However, to understand the preventive effect of L. reuteri and the underlying mechanisms on viral infections, many studies must be performed. Our study used L. reuteri and heat-inactivated L. reuteri to
∗ Corresponding author. College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University, 2888 Xincheng Street, Changchun, 130118, China. ∗∗ Corresponding author. College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University, 2888 Xincheng Street, Changchun, 130118, China. E-mail addresses: [email protected] (C. Wang), [email protected] (G. Yang). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.micpath.2019.103754 Received 19 June 2019; Received in revised form 12 September 2019; Accepted 16 September 2019 Available online 17 September 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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2.5. Cytotoxicity of Lactobacillus reuteri
intervene in C57BL/6 mice and mouse peritoneal macrophages infected with S. typhimurium for the purpose of studying the early interaction in immune system macrophages, pathogens and potential protective probiotics.
To evaluate the cytotoxicity of L. reuteri and heat-inactivated L. reuteri, we examined cell viability using the MTS assay as previously described in Ref. [14]. Peritoneal macrophages (2 × 104 cells per well) were transferred to a 96-well plate and treated with saline and different concentrations of L. reuteri and inactivated L. reuteri (105, 106 and 107 CFU) for 1 h. Subsequently, the above materials were washed with PBS, and the cells were incubated for an additional 24 h at 37 °C. MTS was added to the 96-well plate for further cultivation of 4 h, and the absorbance of each well at 490 nm was measured using a microplate reader. The percentage of cell viability was calculated using the following formula: cell viability (%) = (ODsample – ODblank)/(ODcontrol- ODblank) × 100.
2. Materials and methods 2.1. Reagents Dulbecco's modified Eagle’ medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, phosphate-buffered saline (PBS) and other miscellaneous cell culture reagents were purchased from HyClone Laboratories (Logan, UT, USA). Fluorescent microspheres were obtained from Molecular Probes (Eugene, Oregon, USA). MTS was obtained from Promega (Madison, Wisconsin, USA). A nitrite assay kit was purchased from Beyotime Biochemical Co. (Haimen, China). Antimouse F4/80 (clone BM8) antibodies conjugated to PE were obtained from eBioscience (CA, USA), and anti-mouse CD80 (clone 16-10A1) antibodies conjugated to FITC, anti-mouse CD86 (clone GL1) antibodies conjugated to PE, anti-mouse CD40 (clone 3/23) antibodies conjugated to FITC, and anti-mouse CD11b (clone M1/70) antibodies conjugated to APC from BD Pharmingen (Erembodegem, Belgium) were also procured for flow cytometric analysis. All 96-well, 12-well and 24-well plates were obtained from Corning (USA).
2.6. Flow cytometry Peritoneal macrophages were obtained from the murine abdominal cavity and resuspended to a single cell suspension to assess macrophage purification as previously described [15]. After 24 h of cultivation, peritoneal macrophages were stained with fluorochrome-conjugated antibodies, anti-mouse F4/80-PE and anti-mouse CD11b-APC. Subsequently, the fluorescence in the macrophages was examined by fluorescence-activated cell sorting (FACS) analysis with a BD LSR-FORTESA (BD Biosciences), and the data were analyzed using FlowJo software, version 7.6.0. In a second experiment, peritoneal macrophages were treated with saline, L. reuteri and inactivated L. reuteri. The cell suspensions were stained with fluorescein isothiocyanate anti-mouse CD86-PE, antimouse CD80-FITC, and anti-mouse CD40-FITC antibodies to analyze the activation effects of L. reuteri on macrophages. The detection method and data analysis were the same as those in the above experiment.
2.2. Cells Peritoneal macrophages were obtained from the peritoneal cavity of 6-week-old C57BL/6 mice [12]. After one day, mice were injected with Brewer's thioglycollate broth through intraperitoneal injection. DMEM culture containing 3% FBS was injected into the peritoneal cavity of mice, and the peritoneal cells were obtained along with the medium at 84–120 h, as previously described in Ref. [13]. The obtained peritoneal macrophages were cultivated in DMEM containing fetal bovine serum and penicillin-streptomycin at 37 °C in humidified conditions with 5% CO2, and adherent cells were removed after 2 h.
2.7. Peritoneal macrophages killing intracellular bacteria Extraction of mouse peritoneal macrophages was carried out as previously described [16]. The obtained murine peritoneal macrophages were cultured for 2 h, and nonadherent and dead cells were removed. The macrophages were then plated in a 12-well plate at 2 × 105 cells per well (with antibiotic-free medium) and treated with saline, L. reuteri or inactivated L. reuteri for 1 h (2 × 106 CFU per well). Subsequently, Salmonella was added to the 12-well plate (2 × 106 CFU per well) and incubated at 37 °C for 1 h. The culture medium was discarded, and the plates were washed with sterile PBS. Then, gentamicincontaining DMEM was added to the plate to kill extracellular bacteria. After 1 h of incubation, the gentamicin-containing medium was replaced by antibiotic-free medium, which was cultured for another 2 and 4 h. Macrophages were lysed with 1% Triton X-100 to release intracellular bacteria, and serial dilutions were plated on SalmonellaShigella agar plates. The CFU were enumerated by counting black-centered colonies after incubating the plates for 16–18 h at 37 °C.
2.3. Experimental animal ethical status Six-to eight-week-old female C57BL/6 mice (Beijing HFK Bioscience Co., Ltd., China) were fed a standard mouse chow diet and acclimatized for 7 days prior to the commencement of feeding experiments. All animal experiments were approved by the Animal Care and Ethics Committees of the Jilin Agriculture University, China, and performed according to the Jilin Agriculture University guidelines for Laboratory Animals Care and Usage. The animal facility of the Jilin Agricultural University is fully accredited by the National Association of Laboratory Animal Care. 2.4. Bacterial strain
2.8. Fluorescent microspheres phagocytosis assay L. reuteri ATCC 55730 was obtained from the Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University. L. reuteri was transferred to de Man Rogosa Sharpe (MRS) broth and incubated at 37 °C overnight. Subsequently, bacteria were centrifuged, washed and resuspended in sterile saline, and 1 × 107 CFU of L. reuteri were injected per mouse via the intraperitoneal route. Heatinactivated L. reuteri was boiled for 10 min before use. Salmonella typhimurium ATCC 14028 was obtained from the Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University; it was grown at 37 °C with shaking in Luria Bertani (LB) until the mid-exponential phase (absorbance at 660 nm, 1.0) and then diluted with sterilized saline to a concentration appropriate for each experiment.
Peritoneal macrophages obtained from the peritoneal cavity of mice were transferred to 24-well plates at 2 × 105 cells per well. After 2 h of incubation, nonadherent and dead cells were removed, and peritoneal macrophages were treated with saline, L. reuteri or inactivated L. reuteri for 1 h. Fluorescence microspheres were added to the plates at 2 × 106 per well, and then the plates were cultured for 45 and 90 min. Subsequently, the cells were stained with DAPI in the dark for 15 min and observed under a fluorescence microscope. 2.9. Estimation of NO in the cell culture supernatant Peritoneal macrophages were transferred to a 12-well plate 2
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(2 × 105 cells/well) and treated with saline, L. reuteri or inactivated L. reuteri for 1 h. Subsequently, S. typhimurium (2 × 106 CFU/well) was added to the plate, and the bacteria were removed after 1 h of cultivation and incubated for 2, 4 and 6 h. To estimate the effects of L. reuteri and inactivated L. reuteri on the NO production of peritoneal macrophages, a Nitrate/Nitrite Assay Kit was used to detect nitric oxide levels in the cell supernatant.
These results indicated that different concentrations of L. reuteri and inactivated L. reuteri had no negative effect on cell activity. The multiplicity of infection in subsequent experiments was set at 10 according to this result. 3.2. L. reuteri-induced macrophage activation To detect the purity of macrophages extracted from the abdominal cavity of mice, after removing nonadherent cells and culturing for a period of time, cell surface staining was performed using directly conjugated antibodies: anti-mouse F4/80 and CD11b for purity of peritoneal macrophages and CD40, CD80 and CD86 for L. reuteri-activated macrophages. Flow cytometry results revealed that the peritoneal macrophages we obtained were highly efficient in differentiation (Fig. 2A), and the peritoneal macrophages treated with L. reuteri showed higher expression of CD40 (***P < 0.001), CD80 (***P < 0.001) and CD86 (**P < 0.01) than peritoneal macrophages treated with saline (Fig. 2B and C). However, inactivated L. reuteri enhanced the expression of CD40 (**P < 0.01) and CD86 (*P < 0.05) in macrophages compared with that in the saline group macrophages, which indicated that L. reuteri activated macrophages and that its effect was better than that of inactivated L. reuteri.
2.10. Immunization and infection 24 female C57BL/6 mice were randomly divided into 3 groups, each group consisted of 8 mice, and the immunization procedures were as follows. Two groups of mice were immunized by intraperitoneal injection with L. reuteri or inactivated L. reuteri (1 × 107 CFU) every other day from the first day and then infected with a lethal dose of S. typhimurium (1 × 104 CFU) by intraperitoneal injection on the second day. The procedure of the third group was the same as above; however, mice were injected with the same volume of saline as that for the L. reuteri injection every other day and then infected with S. typhimurium the next day. Weight loss was recorded from the first day, and the survival rate was calculated. 2.11. CFU analysis
3.3. L. reuteri reduces intracellular bacterial survival Liver and spleen tissues were collected 3 days after the mice were challenged with S. typhimurium, and liver and spleen tissues were weighed and homogenized in sterile PBS (g/ml). Appropriate dilutions were plated on Salmonella-Shigella agar plates. The CFU were calculated by counting black-centered colonies after incubating the plates for 16–18 h at 37 °C.
3. Results
Peritoneal macrophages were treated with saline, L. reuteri or inactivated L. reuteri for 1 h, then fluorescence microspheres were added. After culturing for another 45 and 90 min, intracellular fluorescence intensity was observed with a fluorescence microscope (Fig. 3A). The results showed that the ratio of fluorescence microspheres to cells in the L. reuteri and inactivated L. reuteri groups was significantly higher than that in the saline group at 45 min (Fig. 3B). Differences were also observed between the L. reuteri group and the control group after 90 min of treatment; however, there was no difference in the ratio of fluorescence microspheres to cells between the inactivated L. reuteri group and the control group (Fig. 3C), perhaps because the phagocytosis ability increased in the control group over time, while the phagocytosis ability remained unchanged in the L. reuteri and inactivated L. reuteri groups. The results indicated that both L. reuteri and inactivated L. reuteri could enhance peritoneal macrophage phagocytosis, and L. reuteri showed better phagocytosis enhancement activity than inactivated L. reuteri.
3.1. Cytotoxicity of Lactobacillus reuteri
3.4. L. reuteri improved macrophage phagocytosis
To explore the toxic effects of L. reuteri on macrophages, we transferred peritoneal macrophages into 96-well plates with 104 cells per well. Macrophages were infected with different concentrations of L. reuteri and inactivated L. reuteri (105, 106, or 107 CFU per well), and cell viability was detected by MTS assay after infection. The results showed that there was no difference in cell viability among the 105, 106, and 107 CFU of L. reuteri treatment groups and the control group (Fig. 1A). Similarly, the inactivated L. reuteri showed the same results (Fig. 1B).
Peritoneal macrophages isolated from mice were cultured in the presence of L. reuteri, inactivated L. reuteri or saline for 1 h, and the cells were infected with S. typhimurium for 1 h, followed by 2 and 4 h of incubation. Cells were lysed, and the intracellular bacteria were counted. The results showed that there was no significant difference in the number of intracellular bacteria between the saline group and the L. reuteri- or the inactivated L. reuteri-treated group at 2 h. However, the number of intracellular bacteria in the L. reuteri or inactivated L. reuteri
2.12. Statistical analysis Statistical analysis results are expressed as the mean values and standard deviations (SDs) of triplicate experiments. The data were visualized using GraphPad Prism 5 software. The statistical significance was analyzed using an unpaired two-tailed t-test and a one-way ANOVA test, and P-values of less than 0.05 were considered statistically significant.
Fig. 1. Cytotoxicity of Lactobacillus reuteri on peritoneal macrophages. Peritoneal macrophages were transferred to a 96-well plate and treated with saline and different concentrations of L. reuteri and heat-inactivated L. reuteri (105, 106 or 107 CFU) for 1 h. Cells were incubated for another 24 h at 37 °C. Cell viability was detected by MTS assay. (A) Effect of L. reuteri on cell viability. (B) Effect of inactivated L. reuteri on cell viability. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, **P < 0.01).
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Fig. 2. Differentiation rate of peritoneal macrophages and effects of L. reuteri on macrophage activation. Mice were sacrificed, and peritoneal macrophages were obtained with peritoneal exudate. (A) Differentiation rate of peritoneal macrophages. The peritoneal macrophage differentiation rate was analyzed via the markers F4/80 and CD11b and quantified by flow cytometry. (B) Effects of L. reuteri on macrophage activation. Peritoneal macrophages were treated with saline, L. reuteri or inactivated L. reuteri for 1 h, and representative flow cytometric analyses of activated macrophages cells with the markers CD40, CD80 and CD86 were performed. (C) The histogram values are the percentages of activated macrophages cells with the markers CD40, CD80, and CD86. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, **P < 0.01, ***P < 0.001).
inactivated L. reuteri for 1 h and then infected with S. typhimurium (1 × 106 CFU/well) for 1 h, and the cells were cultured for 2, 4 and 6 h. As shown in Fig. 4, nitric oxide content in the S. typhimurium group, L. reuteri plus S. typhimurium group and inactivated L. reuteri plus S. typhimurium group was significantly higher than that in the saline group (***P < 0.001), and furthermore, that in the L. reuteri plus S. typhimurium and inactivated L. reuteri plus S. typhimurium groups was significantly higher than that in the S. typhimurium group (**P < 0.01, *P ＜0.05). The nitric oxide levels of the above three groups decreased
group decreased at 4 h compared with that in the saline group (Fig. 3B). The results indicated that L. reuteri and inactivated L. reuteri enhance the ability of peritoneal macrophages to kill intracellular bacteria. 3.5. Effects of L. reuteri on NO production A nitrate/nitrite kit was used to determine the effect of L. reuteri and inactivated L. reuteri on macrophage nitric oxide production. Peritoneal macrophages (1 × 105 cells/well) were treated with L. reuteri or 4
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The above results indicated that L. reuteri and inactivated L. reuteri could increase the production of nitric oxide in macrophages infected with S. typhimurium, thus affecting the antimicrobial ability of macrophages. However, when cells were treated with L. reuteri or inactivated
with time. In contrast, the nitric oxide content in the L. reuteri and inactivated L. reuteri groups showed no difference from that in the saline group in the first 4 h but increased at 6 h, while the nitric oxide content in the saline group remained unchanged. 5
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Fig. 3. L. reuteri and inactivated L. reuteri can improve the phagocytosis and sterilization abilities of peritoneal macrophages. (A) L. reuteri and inactivated L. reuteri improve peritoneal macrophage phagocytosis. Peritoneal macrophages were pretreated with saline, L. reuteri or inactivated L. reuteri for 1 h. Fluorescent microspheres were added to the cells and cultured for 45 or 90 min, and macrophage phagocytosis of the microspheres was observed under a fluorescence microscope. (B) Higher fluorescence of microspheres per cell was observed in the L. reuteri group (n = 20) and inactivated L. reuteri group (n = 22) than in the control group (n = 25) after 45 min of treatment (***P < 0.001). (C) After 90 min of treatment, differences were also observed between the L. reuteri (n = 24) and control groups (n = 18), and no differences were detected between the inactivated L. reuteri group (n = 24) and the control group (**P < 0.01). (D) Intracellular bacteria enumeration in macrophages. Peritoneal macrophages treated as described above were infected with S. typhimurium (1 × 105 CFU) for 1 h, culture medium containing gentamicin was used to kill extracellular bacteria, and intracellular bacteria were counted at the indicated times. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05).
saline group (***P < 0.001), while there was no difference between the L. reuteri and inactivated L. reuteri groups (Fig. 5C). Consistently, spleen S. typhimurium CFU in the L. reuteri and inactivated L. reuteri groups were also significantly lower than those in the saline group (***P < 0.001). However, no difference was observed in S. typhimurium CFU between the L. reuteri group and inactivated L. reuteri group (Fig. 5D). Moreover, there was no difference in the spleen S. typhimurium CFU between the L. reuteri group and the inactivated L. reuteri group. The results indicated that L. reuteri and inactivated L. reuteri inhibited the dissemination of S. typhimurium from the abdominal cavity to the spleen and liver. 4. Discussion Fig. 4. Effects of L. reuteri and inactivated L. reuteri on NO production of macrophages infected with S. typhimurium. Murine peritoneal macrophages (1 × 105 cells/well) were treated with saline, L. reuteri or inactivated L. reuteri (1 × 106 CFU/well) for 1 h. Then, S. typhimurium (1 × 106 CFU/well) was added to peritoneal macrophages and incubated for 1 h. After an additional 2 or 4 h of incubation, the cell culture supernatant was collected, and the NO content in the supernatant was determined. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, **P < 0.01, ***P < 0.001).
Lactobacillus, as a probiotic, interacts with intestinal microbial communities and hosts and has a positive impact on individual health when ingested in sufficient quantities [17]. Lactobacillus reuteri is a widely studied probiotic naturally existing in the digestive tract of most vertebrates and mammals [18]. Lactobacillus reuteri can control bodily endotoxin levels, protect the liver and improve liver detoxification, indicating that Lactobacillus reuteri plays the role of immune regulation in the abdominal cavity, including stimulating peritoneal macrophage activation and inducing antibodies and interferon production [19,20]. Nonactivated macrophages express only a small amount of MHCII and do not express costimulatory molecules. However, macrophages are induced to express MHCII and costimulatory molecules, such as CD80 and CD86, and release inflammatory mediators, promoting the removal of alien components in cells after activation [21]. In this study, we demonstrated the protective effect of L. reuteri on S. typhimurium infection. After treatment with L. reuteri and inactivated L. reuteri, the expression of CD40, CD80 and CD86 on peritoneal macrophages was increased (Fig. 2B), indicating that both L. reuteri and inactivated L. reuteri treatment activate macrophages. Activated macrophages have the functions of releasing inflammatory mediators, presenting antigens and initiating the adaptive immune response and are an important line of defense for the body against foreign substances [22]. Macrophages are important for controlling and removing Salmonella from host cells [23]. When the pathogen invades, some highly conserved pathogen-associated molecular patterns (PAMPs) on pathogens are recognized by pattern recognition receptors (PRRs) on the surface of macrophages, which then absorb into the cell to form phagosomes that are further fused with intracellular lysosomes to form phagocytic lysosomes and digest and remove pathogens through oxygen-dependent and oxygen-independent pathways. Our results suggested that L. reuteri and inactivated L. reuteri treatment enhance macrophage phagocytic activity to kill intracellular pathogens (Fig. 3A and B). Previous studies have also confirmed that Lactobacillus can activate macrophages, enhance phagocytosis ability and inhibit intracellular pathogen survival, thus playing a role in resisting pathogens [24,25]. Three groups of mice were immunized with L. reuteri, inactivated L. reuteri or the same volume of saline by intraperitoneal injection. The mice were infected with S. typhimurium through intraperitoneal injection, and their weight and survival rate were continuously recorded. Compared with the saline group, the L. reuteri and inactivated L. reuteri groups exhibited a significantly improved survival rate and prolonged
L. reuteri alone, the production of nitric oxide in cells was not affected in a short time period, and the production of nitric oxide was increased in a long time period. 3.6. Effects of L. reuteri on weight loss and mortality of infected mice Three groups of mice were treated with saline, L. reuteri or inactivated L. reuteri (106 CFU) by intraperitoneal injection every other day. On the day after the initial immunization, mice were infected with S. typhimurium (104 CFU) by intraperitoneal injection, and the body weight and mortality were recorded for 25 consecutive days. The mice in all three groups exhibited weight loss after S. typhimurium challenge. The L. reuteri group showed slight weight loss and recovered after 5 days (Fig. 5A). Not surprisingly, the saline group succumbed to the lethal challenge of the S. typhimurium challenge within 5 days. However, only one mouse died in the inactivated L. reuteri group on day 5, and all mice in the inactivated L. reuteri group died by day 11. Interestingly, mice in the L. reuteri group began to die after all the mice in the saline group had died, and one of them survived until the end of observation (Fig. 5B). These results effectively demonstrate the potential of L. reuteri and inactivated L. reuteri in protecting mice against S. typhimurium infection, and the protective effect of L. reuteri was superior to that of inactivated L. reuteri. 3.7. Salmonella typhimurium CFU in the liver and spleen Mice were sacrificed 3 days after S. typhimurium challenge. The liver and spleen of each mouse were weighed and homogenized with PBS at a ratio of g of tissue/ml of PBS. Tissue homogenate was plated on Salmonella-Shigella agar plates after appropriate dilution, and bacterial CFU were counted after overnight culture. An approximately ten-fold decrease was observed in the liver S. typhimurium CFU of the L. reuteri group and the inactivated L. reuteri group compared with those of the 6
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Fig. 5. L. reuteri pretreatment protects mice against salmonella infection and reduces the dissemination of salmonella from the abdomen to the liver and spleen. Three groups of mice were pretreated with L. reuteri, inactivated L. reuteri or saline by intraperitoneal injection and subsequently challenged with S. typhimurium. (A) Weight loss was continuously recorded for 25 days. *Statistically significant difference relative to the inactivated L. reuteri group (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Survival rates were continuously recorded for 25 days. *Statistically significant difference relative to the saline group (**P < 0.01). (C, D) Mice were sacrificed at a designated time after challenge with S. typhimurium, the liver and spleen were collected, and the bacterial CFU of per gram of liver and spleen was counted. The results are presented as the means ± SDs of three independent experiments. *Significant differences compared to the saline group (*P < 0.05, ***P < 0.001).
increased with the prolongment of infection. L. reuteri is an indigenous flora constituent in mammals that can form biological barriers in the intestinal tract and other mucosal sites to resist the invasion of pathogenic bacteria and enhance body immunity. In addition, studies have shown that L. reuteri can reduce the colonization of pathogenic bacteria in organs [34]. In this study, similar results showed that L. reuteri could reduce the colonization of S. typhimurium in the mouse liver and spleen. Macrophages induce the production of nitric oxide synthase during the inflammatory reaction, leading to an increase in NO after infection [35]. NO and ROS are considered the second barrier of the innate defense mechanism. NO plays a variety of roles in the innate immune system, such as killing virus-infected cells, tumor cells and parasitic pathogens. In some viral infections, the antiviral effect of NO has been well demonstrated [36]. However, excessive NO can damage DNA, proteins and lipids in cells and tissues. Therefore, the ultimate effect of NO should be considered as a balance between antiviral and cytotoxic effects [37,38]. In short, our results suggested that L. reuteri may play an antibacterial role by enhancing the production of nitric oxide in macrophages (Fig. 4). Nitric oxide, as an reactive nitrogen intermediate, is closely related to reactive oxygen species and other cytokines, and we may conduct further studies in this regard. In conclusion, our experiments showed that L. reuteri and inactivated L. reuteri can activate macrophages, enhance their abilities to phagocytose and to kill intracellular bacteria, and increase the secretion of nitric oxide in macrophages infected with S. typhimurium. In vivo experiments confirmed that L. reuteri and inactivated L. reuteri have
survival time in mice (Fig. 5A and B). However, the protective effect of L. reuteri on mice and peritoneal macrophages was superior to that of inactivated L. reuteri, which may be due to metabolites with antibacterial activity secreted by L. reuteri, such as reuterin and histamine [26,27]. Studies have shown that L. reuteri ameliorates viral infection by regulating the microbial community and secreting metabolites with antiviral components [28]. Reuterin is a nonprotein-based broad-spectrum antimicrobial substance that can widely inhibit the growth of gram-positive bacteria, gram-negative bacteria, yeast, fungi and pathogens [29]. Previous studies have confirmed that the culture supernatant of Lactobacillus upregulated the production of macrophage NO and partially upregulated several inflammatory cytokines in macrophages [30]. In addition, mice in the inactivated L. reuteri group had a longer survival time than those in the control group, although its protective effect on mice was less than that of live L. reuteri, which indicated that the structure of L. reuteri or proteins such as peptidoglycan may have a protective effect. Recent studies have found that L. reuteri stimulates immunomodulatory responses through macrophages and reduces the secretion of proinflammatory factors [31,32]. The liver is an important organ of the body, and its metabolism is related to the gastrointestinal tract and even the whole biological process. It is also an important target of many drugs, oxidation and pathogen invasion [33]. Liver injury will seriously affect the metabolism, detoxification, immune response and antibacterial ability of the body. In our study, the liver and spleen of the mice infected with S. typhimurium were affected, and the number of S. typhimurium in tissues 7
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protective effects on mice infected with S. typhimurium and can reduce the bacterial load in the spleen and liver of mice. In addition, in the above experiments, the protective effect of L. reuteri was superior to that of inactivated L. reuteri, which may be related to bacteriocins, such as reuterin, and other substances produced by L. reuteri.
[13]
[14]
Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
[15]
[16]
Author contributions
[17]
Wentao Yang and Chunfeng Wang conceived and supervised the study; Pingping Jiang and Wentao Yang designed experiments; Pingping Jiang, Wentao Yang, Haibin Huang and Chunwei Shi performed the experiments; Pingping Jiang, Yubei Jin, Yanlong Jiang, Jianzhong Wang, Yuanhuan Kang, Guilian Yang analyzed the data. Pingping Jiang, Wentao Yang and Chunfeng Wang Wrote the paper. All authors reviewed the results and approved the final version of the manuscript.
[18]
[19]
[20]
Funding This work was supported by the National Key Research and Development Program of China (2017YFD0501000, 2017YFD0500400), National Natural Science Foundation of China (31672528), Science and Technology Development Program of Jilin Province (2017YFD0501200, 20170204034NY, 20180520037JH), the Key Funds for Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture (ZJK201808).
[21] [22]
[23]
[24]
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