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In this study, we investigated the effects of linoleic acid (LA) treatment on Brucella abortus infection in professional phagocyte RAW264.7 cells, particularly during the pathogen’s invasion and intracellular growth in these cells, as well as in murine model BALB/c mice focusing on bacterial splenic proliferation and immunoregulatory activities. LA inhibited the growth of Brucella in a doseand time-dependent manner. The ability of the pathogen to enter the phagocytes was inhibited as was its survival within these cells. This was accompanied by increased nitrite accumulation in these cells at 24 h post-infection. The concentration of LA used in the present study did not affect the total body weight or liver function of the mice. During Brucella infection, the total splenic weight of these animals was not changed; rather, resistance to bacterial proliferation was enhanced in the spleen. Furthermore, mice treated with LA displayed elevated levels of IL-12 and IFN-γ but reduced levels of IL-10 during infection. The findings in this study showed the regulatory role of LA against B. abortus infection suggesting its potential use in designing intervention strategy for brucellosis.

We previously identified β-sitosterol (BS) as one of the most abundant compounds found in Korean red ginseng oil. BS is a widely prevalent vegetable-derived phytosterol with many known health benefits. Here, we investigated the efficacy of BS against Brucella (B.) abortus infection. BS showed no effect on bacterial growth but attenuated internalization, intracellular survival and MAPKs-linked intracellular signaling in RAW264.7 cells. BS treatment in cells is also associated with increased nitrite concentration during infection at 24 h. Slightly enhanced resistance to B. abortus infection was observed in mice orally given BS, which could be mediated by induced production of proinflammatory cytokines. Taken together, our study demonstrates the contribution of BS treatment against B. abortus infection although further investigation is encouraged to maximize its beneficial effects against intracellular infection.

Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely outer membrane protein (Omp) 16, Omp19, Omp28, and 50S ribosomal protein L7/L12 was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. The immunoreactivity of these four recombinant proteins as well as pCold-TF vector reacted with Brucella-positive serum individually, but not with Brucella-negative serum by immunoblotting assay. CSV-treated RAW 264.7 cells significantly induced production of IFN-γ and IL-12 while decreased IL-10 production at the late stage of infection compared to PBS-treated control cells. In addition, the enhancement of nitric oxide production together with cytokines secretion profile in CSV-treated cells proved that CSV notably activated bactericidal mechanisms in macrophages. Consistently, mice immunized with CSV strongly elicited production of pro-inflammatory cytokines TNF-α, IL-6 and MCP-1 compared to PBS control group. Moreover, the concentration of IFN-γ was >IL-10 and titers of IgG2a were also heightened compared to IgG1 in CSV-immunized mice which suggest that CSV induced predominantly T helper 1 T cell. These results suggest that the CSV used in the present study is a potential candidate as a preventive therapy against brucellosis.

Background We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. Objectives We aimed to investigate the efficacy of PA against B. abortus infection using a murine cell line and a murine model. Methods Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O₂⁻) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. Results PA did not affect the growth of B. abortus. PA treatment in macrophages did not change B. abortus uptake but it did attenuate the intracellular survivability of B. abortus. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression. Compared to control cells, reduced nitrite accumulation, augmented O₂⁻, and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus-infected cells. Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. Conclusions Our results suggest that PA participates in the control of B. abortus within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus infection by fatty acids.

Chronic infection with intracellular Brucella abortus (B. abortus) in livestock remains as a major problem worldwide. Thus, the search for an ideal vaccine is still ongoing. In this study, we evaluated the protective efficacy of a combination of B. abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12) and malate dehydrogenase (rMDH), cloned and expressed into a pMal vector system and DH5α, respectively, and further purified and applied intraperitoneally into BALB/c mice. After first immunization and two boosters, mice were infected intraperitoneally (IP) with 5 × 104 CFU of virulent B. abortus 544. Spleens were harvested and bacterial loads were evaluated at two weeks post-infection. Results revealed that this combination showed significant reduction in bacterial colonization in the spleen with a log protection unit of 1.31, which is comparable to the average protection conferred by the widely used live attenuated vaccine RB51. Cytokine analysis exhibited enhancement of cell-mediated immune response as IFN-γ is significantly elevated while IL-10, which is considered beneficial to the pathogen's survival, was reduced compared to control group. Furthermore, both titers of IgG1 and IgG2a were significantly elevated at three and four-week time points from first immunization. In summary, our in vivo data revealed that vaccination with a combination of five different proteins conferred a heightened host response to Brucella infection through cell-mediated immunity which is desirable in the control of intracellular pathogens. Thus, this combination might be considered for further improvement as a potential candidate vaccine against Brucella infection.

The aim of this work is to investigate the protective efficacy of emodin, an active, naturally-occurring anthraquinone derivative of several traditional Chinese herbs, against Brucella abortus infection in macrophages. Brucella were incubated with different concentrations of emodin and showed that bacterial survival rates were markedly reduced in a dose-dependent manner at increasing incubation time points. Through bacterial infection assay, the highest non-cytotoxic concentration of emodin demonstrated attenuated invasion of Brucella into macrophages, however it did not inhibit the growth of these pathogens within the host cells. On the other hand, emodin effectively decreased the number of bacteria that adhered to host cells, which indicated its potential as an anti-adhesin agent. Furthermore, using immunoblotting and FACS assay for detecting MAPK signaling proteins and F-actin polymerization, respectively, the results showed that the emodin-incubated cells displayed modest reduction in the phosphorylation levels of ERK1/2 and inhibition of F-actin polymerization as compared to control cells. These findings indicate the potential use of emodin as a naturally-occurring alternative method for the prevention of animal brucellosis although this requires confirmation of safe clinical doses.

In this study, we investigated the effects of gallic acid (GA) in intracellular signaling within murine macrophages and its contribution to host immunity during Brucella infection. In vitro analysis revealed that GA treatment decreased F-actin content and suppressed p38α phosphorylation level. In vivo analysis showed that GA treatment reduced inflammation and proliferation of Brucella in spleens of mice in comparison to PBS treatment yielding a significant protection unit. For the analysis of immune response, the uninfected GA-treated mice showed increased production of IFN-γ and MCP-1, and the Brucella-infected GA-treated mice showed elevated levels of IL-12p70, TNF, IFN-γ, MCP-1, IL-10 and IL-6 in comparison to negative and positive control groups, respectively. These findings demonstrate the therapeutic effects of GA against Brucella infection through interference on intracellular signaling pathway, induction of cytokine production and protection from bacterial proliferation in spleens of mice.

Brucella abortus, one of the most important members of the genus Brucella responsible for human disease, is an intracellular pathogen capable of avoiding or interfering components of the host immune responses that are critical for its virulence. GPR84, on the other hand, is a seven-transmembrane GPCR involved in the inflammatory response and its induced expression was associated with B. abortus infection of RAW264.7 cells. Here we examined the effects of the reported GPR84 surrogate and endogenous agonists, namely 6-n-octylaminouracil (6-OAU) and lauric acid (LU), respectively in the progression of B. abortus infection in a cell and mouse models. The in vitro studies revealed the LU had bactericidal effect against Brucella starting at 24 h post-incubation. Adhesion of Brucella to RAW264.7 cells was attenuated in both 6-OAU and LU treatments. Brucella uptake was observed to be inhibited in a dose and time-dependent manner in 6-OAU but only at the highest non-cytotoxic concentration in LU-treated cells. However, survival of Brucella within the cells was reduced only in LU-treated cells. We also investigated the possible inhibitory effects of the agonist in other Gram-negative bacterium, Salmonella Typhimurium and we found that both adhesion and uptake were inhibited in 6-OAU treatment and only the intracellular survival for LU treatment. Furthermore, 6-OAU treatment reduced ERK phosphorylation and MCP-1 secretion during Brucella infection as well as reduced MALT1 protein expression and ROS production in cells without infection. LU treatment attenuated ERK and JNK phosphorylation, MCP-1 secretion and NO accumulation but increased ROS production during infection, and similar pattern with MALT1 protein expression. The in vivo studies showed that both treatments via oral route augmented resistance to Brucella infection but more pronounced with 6-AOU as observed with reduced bacterial proliferation in spleens and livers. At 7 d post-treatment and 14 d post-infection, 6-OAU-treated mice displayed reduced IFN-γ serum level. At 7 d post-infection, high serum level of MCP-1 was observed in both treatments with the addition of TNF-α in LU group. IL-6 was increased in both treatments at 14 d post-infection with higher TNF-α, MCP-1 and IL-10 in LU group. Taken together, 6-OAU and LU are potential candidates representing pharmaceutical strategy against brucellosis and possibly other intracellular pathogens or inflammatory diseases.

In this study, we evaluated the inhibitory effect of a rice bran mixture extract (RBE) on Brucella abortus pathogenesis in professional (RAW 264.7) and nonprofessional (HeLa) phagocytes. We fermented the rice bran mixture and then extracted it with 50% ethanol followed by gas chromatography-mass spectrometry to identify the components in RBE. Our results clearly showed that RBE caused a significant reduction in the adherence of B. abortus in both cell lines. Furthermore, analysis of phagocytic signaling proteins by western blot assay revealed that RBE pretreatment resulted in inhibition of phosphorylation of JNK, ERK, and p38, leading to decline of internalization compared with the controls. Additionally, the intensity of F-actin observed by fluorescence microscopy and FACS was remarkably reduced in RBE-pretreated cells compared with control cells. However, the intracellular replication of B. abortus within phagocytes was not affected by RBE. Taken together, these findings suggest that the phagocytic receptor blocking and suppressive effects of RBE on the MAPK-linked phagocytic signaling pathway could negatively affect the invasion of B. abortus into phagocytes.

In this study, we investigated the protective effects of tannin-derived components, gallic acid (GA) and tannic acid (TA), in vitro and in vivo against Salmonella infection in mice. Both GA and TA showed antibacterial effects against Salmonella (S.) Typhimurium as well as inhibitory effects on the adherence, invasion, and intracellular growth of the pathogens in macrophages. Following a lethal dose of Salmonella infection in mice, reduced virulence in both GA- and TA-treated groups was observed based on reduced mortality rates. In the non-infected groups, the average weights of the spleens and livers of GA- or TA-treated mice were not significantly different with the control group. In addition, the average weights of these organs in all of the Salmonella-infected groups were not significantly different but the numbers of bacteria in the spleens and livers in both GA- and TA-treated mice were significantly reduced. The levels of cytokine production in non-infected mice revealed that GA-treated and TA-treated mice elicited an increased level of IFN-γ, and both IFN-γ and MCP-1, respectively, as compared with the PBS-treated group. These findings highlight the potential of GA and TA as alternatives for the treatment of salmonellosis and as supplements to conventional antimicrobial food additives.

To date, the implications of interleukin 6 (IL-6) for immune responses in the context of Brucella infection are still unknown. In the present study, we found that Brucella abortus infection induced marked production of IL-6 in mice that was important for sufficient differentiation of CD8+ T cells, a key factor in Brucella clearance. Blocking IL-6 signaling also significantly induced serum IL-4 and IL-10, together with a decreased gamma interferon (IFN-γ) level, suggesting that IL-6 is essential for priming the T-helper (Th) 1 cell immune response during Brucella infection. The IL-6 pathway also activated the bactericidal activity of primary and cultured macrophages. Bacterial killing was markedly abrogated when IL-6 signaling was suppressed, and this phenomenon was mainly associated with decreased activity of lysosome-mediated killing. Interestingly, suppressor of cytokine signaling 3 (SOCS3) was important for regulating the IL-6-dependent anti-Brucella activity through the JAK/STAT pathway. During early infection, in the absence of SOCS3, IL-6 exhibited anti-inflammatory effects and lysosome-mediated killing inhibition; however, the increase in SOCS3 successfully shifted functional IL-6 toward proinflammatory brucellacidal activity in the late stage. Our data clearly indicate that IL-6 contributes to host resistance against B. abortus infection by controlling brucellacidal activity in macrophages and priming cellular immune responses.

To date, the antimicrobial activity of arachidonic acid (AA) with regard to pathogenesis of Brucella in macrophages is unknown. We found that AA is highly toxic to B. abortus in a time- and dose-dependent manner. Transcription profiling of different groups of phospholipases A2 (PLA2) was examined, ten PLA2 were detected including cPLA2-IV-A, cPLA2-IV-B, iPLA2-VI, sPLA2-I-B, sPLA2-II-C, sPLA2-II-D, sPLA2-II-E, sPLA2-V, sPLA2-X, sPLA2-XII-A. Phagocytic signaling investigation indicated that AA treatment attenuated p38α activity in infected culture macrophages possibly leading to inhibition of Brucella internalization. Post-treatment with the fatty acid did not influence bacterial intracellular multiplication or alter production of antimicrobial effectors like ROS and NO in RAW 264.7 cells. On the other hand, AA administration significantly reduced bacterial load and modestly inhibited pro-inflammatory cytokine secretion including TNF, IFN-γ and IL-6 in mice plasma. To our knowledge, we are the first to suggest that B. abortus invasion to RAW 264.7 macrophages is impaired by AA.

Brucellosis is one of the most important and widespread zoonosis worldwide responsible for serious economic losses and considerable public health burden. In this study, we investigated the modulatory effect of a microtubule-inhibitor, nocodazole, on B. abortus infection in murine macrophages and in a mouse model. Nocodazole activated macrophages and directly inhibited the growth of Brucella in a dose-dependent manner. Nocodazole increased adhesion but reduced invasion and intracellular growth of Brucella in macrophages although it did not affect co-localization of Brucella with LAMP-1. In addition, nocodazole negatively affected actin polymerization, and weakly activated ERK and p38α but significantly activated JNK in non-infected cells. After subsequent infection, nocodazole weakly inhibited activation of ERK and p38α. For the in vivo tests, nocodazole -treated mice displayed elevated levels of IFN-γ, MCP-1 and IL-10 while Brucella-infected nocodazole -treated mice showed high levels of TNF, IFN-γ, MCP-1, IL-10 and IL-6 as compared to controls. Furthermore, nocodazole treatment reduced inflammation and Brucella proliferation in the spleens of mice. These findings highlight the potential use of nocodazole for the control of brucellosis although further investigations are encouraged to validate its therapeutic use in animal hosts.

To date, the implications of prostaglandin I2 (PGI2), a prominent lipid mediator for modulation of immune responses, has not been clearly understood in Brucella infection. In this study, we found that cyclooxygenase-2 (COX-2) was significantly expressed in both infected bone marrow-derived macrophages (BMMs) and RAW 264.7 cells. Prostaglandin I2 synthase (PTGIS) expression was not significantly changed, and PGI2 receptor (PTGIR) expression was downregulated in BMMs but upregulated in RAW 264.7 macrophages at late infection. Here, we presented that PGI2, a COX-derived metabolite, was produced by macrophages during Brucella infection and its production was regulated by COX-2 and IL-10. We suggested that PGI2 and selexipag, a potent PGI2 analogue, inhibited Brucella internalization through IP signaling which led to down-regulation of F-actin polymerization and p38α MAPK activity. Administration with selexipag suppressed immune responses and resulted in a notable reduction in bacterial burden in spleen of Brucella-challenged mice. Taken together, our study is the first to characterize PGI2 synthesis and its effect in evasion strategy of macrophages against Brucella infection.

In this study, two recombinant proteins encoded by Brucella abortus genes Adk and SecB were evaluated as single subunit vaccine (SSV) as well as combined subunit vaccine (CSV) against B. abortus infection in BALB/c mice. These genes were cloned into pcold-TF expression system and recombinant proteins were expressed in Escherichia coli DH5α. The immunoreactivity of purified rAdk and rSecB was analyzed by immunoblotting showing that purified rAdk and rSecB as well as pcold-TF vector strongly reacted with Brucella-positive serum. Mice were immunized intraperitoneally with SSVs, CSV, pcold-TF, RB51 and PBS. The analysis of cytokine revealed that SSVs and CSV can strongly induce production of proinflammatory cytokines TNF and IL-6, suggesting that these subunit vaccines elicited innate immune response, particularly, activated antimicrobial mechanism of macrophages to limit the initial infection. On the other hand, immunization with SSVs and CSV elicited strong IFN-γ production and decreased IL-10 production compared to PBS group. The secretion profiles of IFN-γ and IL-10 together with an enhancement of blood CD4+ population and significantly induced specific IgG1 and IgG2a antibodies indicated that SSVs and CSV induced not only humoral immunity but also T helper 1 T cell immunity. Finally, spleen proliferation and bacterial burden in the spleen of mice vaccinated with these subunit vaccines were significantly lower than those of PBS group, which conferred significant protection against B. abortus infection. Altogether, the potential of these antigens of B. abortus could be prospective candidates to develop subunit vaccines against brucellosis.

Brucella as a stealthy intracellular pathogen avoids activation of innate immune response. Here we investigated the contribution of an adenosine receptor, Adora2b, during Brucella infection in professional phagocyte RAW 264.7 cells and in a murine model. Adora2b-deficient cells showed attenuated Brucella internalization and intracellular survival with enhanced release of IL-6, TNF-α, IL-12 and MCP-1. In addition, blockade of Adora2b using MRS 1754 treatment in mice resulted in increased total weight of the spleens but suppressed bacterial burden in these organs accompanied by elevated levels of IL-6, IFN-γ, TNF-α, IL-12 and MCP-1, while reduced IL-10. Overall, we proposed that the Adora2b participates in the successful phagocytic pathway and intracellular survival of Brucella in RAW 264.7 cells, and could be a potential therapeutic target for the treatment of acute brucellosis in animals.

The bacterial lipopolysaccharide (LPS) mainly contributes to the structural integrity, survival and protection barrier against harsh environments. Therefore, the early stages in LPS or lipid A biosynthesis are attractive targets in the identification and development of inhibitors which would be effective against infections caused by Gram-negative bacteria. The bacterial outer membrane proteins (OMPs) meanwhile function as maintenance for structure, adhesion to other cells and substances, as well as development of resistance to antimicrobials. The LPS and LPS-related molecules, and OMPs are important immunogenic components of several important pathogens including Brucella, which have been extensively used in immunological studies and in the diagnosis of diseases. Here we review the importance, structure, functions and immunogenic aspects of LPS and OMPs particularly of Brucella which can be targeted for the prevention and diagnosis of brucellosis.

Background: Modulation of gene expression is a fundamental requirement for adaptation of intracellular Brucella abortus. Since most of the current understanding mainly focus on professional phagocytes and renal involvement is uncommon in brucellosis, our aim was to identify and analyze changes in B. abortus gene expression in response to intracellular environment within a bovine kidney cell line. Methodology: B. abortus RNA were isolated from Madin-Darby bovine kidney (MDBK) epithelial cells during replicative phase and the transcriptional profile of intracellular B. abortus was characterized using microarray analysis. Results and interpretation: The microarray analysis revealed a total of 1,623 differentially expressed genes of ≥ 2-fold–788 (25.44%, 788/3098) upregulated and 835 (26.95%,835/3098) down-regulated genes as compared with free-living Brucella. Among these identified genes, 81 and 185 were upregulated and down-regulated at ≥ 7-fold, respectively, showing a marked induction of genes involved in transcription and distinct repression of genes involved in translation, ribosomal structure and biosynthesis. Conclusion: The identified genes in this study may provide new insights into the molecular interactions between B. abortus and non-phagocytic bovine cell line, MDBK. In addition, several differentially highly expressed transcripts were hypothetical genes with unknown function and/or unclassified which require further characterization due to their potential contribution in the virulence and strategy of Brucella to survive and proliferate within the host.

In this study, we assessed the protective efficacy of single subunit vaccines, encoded by the B. abortus 544 genes aspC, dps, yaeC and inpB, against B. abortus infection in mice. First, immunization with these antigens, with the exception of the YaeC protein, was found to elicit both humoral and cellular immune responses with IgG2a being dominant over IgG1. In addition, a massive production of IFN-γ but lower degree of IL-10 was observed, suggesting that all three antigens were able to induce predominantly cell-mediated immunity in response to B. abortus infection. Further investigation of a combined subunit vaccine (CSV) consisting of purified AspC, Dps, InpB and Ndk proteins showed a superior protective effect in mice against brucellosis. The intraperitoneal injection of this combination was shown to induce a remarkable production of IFN-γ and IL-2, which occurred in conjunction with an increase of blood CD4+ and CD8+ T cell proportions. In addition, the higher titer of IgG2a compared to IgG1 elicited by this CSV was obtained, suggesting that this CSV induced a typical T-helper-1-dominated immune response in vivo. Furthermore, the protection level induced by this combination was significantly higher than that induced by single antigens and was not significantly different compared to a group immunized with a live attenuated vaccine (RB51). Altogether, our findings suggest that the combination of different immunogenic antigens could be a useful approach for the development of a new, effective and safe brucellosis vaccine that can replace current vaccine strains.

Lipocalin 2 (Lcn2) is an important innate immunity component against bacterial pathogens. In this study, we report that Lcn2 is induced by Brucella (B.) abortus infection and significantly contributes to the restriction of intracellular survival of Brucella in macrophages. We found that Lcn2 prevented iron uptake by B. abortus through two distinct mechanisms. First, Lcn2 is secreted to capture bacterial siderophore(s) and abrogate iron import by Brucella. Second, Lcn2 decreases the intracellular iron levels during Brucella infection, which probably deprives the invading Brucella of the iron source needed for growth. Suppression of Lcn2 signalling resulted in a marked induction of anti-inflammatory cytokine, interleukin 10, which was shown to play a major role in Lcn2-induced antibrucella immunity. Similarly, interleukin 6 was also found to be increased when Lcn2 signalling is abrogated; however, this induction was thought to be an alternative pathway that rescues the cell from infection when the effective Lnc2 pathway is repressed. Furthermore, Lcn2 deficiency also caused a marked decrease in brucellacidal effectors, such as reactive oxygen species and nitric oxide but not the phagolysosome fusion. Taken together, our results indicate that Lcn2 is required for the efficient restriction of intracellular B. abortus growth that is through limiting iron acquisition and shifting cells to pro-inflammatory brucellacidal activity in murine macrophages.

We investigated the involvement of chemokine receptor type 4 (CXCR4) signaling on the outcome of Brucella (B.) abortus 544 infection in murine macrophages and in a mouse model. CXCR4 manipulation were first evaluated for Brucella invasion and intracellular survival efficiency, mitogen-activated protein kinases (ERK1/2, JNK, p38α) activation and generation of nitric oxide (NO), and then in the splenic bacterial proliferation and cytokine production in BALB/c mice. CXCR4 blockade is involved in the successful control of Brucella invasion, reduction of ERK1/2 phosphorylation and inhibition of nitric oxide release from macrophages. Furthermore, using a reported CXCR4-specific antagonist AMD3100 resulted in splenomegaly but attenuated Brucella proliferation in these organs with elevated serum levels of MCP-1, TNF and IL-12. These findings provide insights on the contribution of CXCR4 signaling in the phagocytic pathway and immune modulation during B. abortus infection.