Prostaglandin I2 (PGI2) inhibits Brucella abortus internalization in macrophages via PGI2 receptor signaling, and its analogue affects immune response and disease outcome in mice.

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 PGI2receptor (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.

Transcriptomic profiling of phospholipase A2 and the role of arachidonic acid during Brucella abortus 544 infection in both in vitro and in vivo systems.

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.

Immunogenicity and protective response induced by recombinant Brucella abortus proteins Adk, SecB and combination of these two recombinant proteins against a virulent strain B. abortus 544 infection in BALB/c mice.

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.

The immunomodulatory effect of antimicrobial peptide HPA3P restricts Brucella abortus 544 infection in BALB/c mice.

The discovery of antimicrobial peptides (AMPs) in recent years has been promising for the treatment of multidrug resistant pathogenic microbes. Brucellosis is still considered one of the most common zoonoses in the world. In this study, we evaluated the effect HPA3P peptide in the bacterial uptake and intracellular growth of Brucella abortus (B. abortus) 544 in murine macrophages RAW 264.7. HPA3P was further utilized in a mouse model for infection and treatment. This peptide did not show cytotoxicity or bactericidal effect to B. abortus. However, it inhibited bacterial internalization at 0, 15 and 30 min incubation at two different doses at 12 and 24 µM as well as reduced intracellular growth after 2, 24 and 48 h incubation. Mice treated with HPA3P demonstrated a significant 1.01-log reduction (P < 0.0001) and spleen weight reduction compared to the nanocarrier control (P < 0.01). Significant increases in key cytokines Interferon-γ (IFN-γ) and Tumor necrosis factor (TNF) at 3, 7 and 14 days post-infection were observed in HPA3P treated mice similar to the antibiotic control group with both compared to the nanocarrier control. Monocyte chemoattractant protein-1 (MCP-1) was also heightened at 14 days post-infection. Histopathological analysis also suggests reduced bacterial granuloma in the liver and spleens of HPA3P treated group compared with the nanocarrier control group. In this study, the modulation of crucial cytokines IFN-γ and TNF might have led to a considerable reduction in the proliferation of B. abortus in a mouse model of brucellosis. Further investigation might be required to maximize the efficacy of HPA3P treatment in murine brucellosis.

Evaluation of the combined use of the recombinant Brucella abortus Omp10, Omp19 and Omp28 proteins for the clinical diagnosis of bovine brucellosis.

Currently, there are several serodiagnostic tools available for brucellosis, however, it is difficult to differentiate an active infection from vaccination. Hence, there is a great need to develop alternative means that can distinguish between these two conditions without utilizing lipopolysaccharide (LPS). This study was an attempt to determine the efficacy of combined recombinant Brucella (B.) abortus outer membrane proteins (rOmps) and individual rOmps in the serodiagnosis of brucellosis by enzyme linked immunosorbent assay (ELISA), utilizing both that standard tube agglutination test (TAT)-positive and -negative serum samples from Korean native cattle. The results are very interesting and promising because the combined rOmp antigens used in the study were highly reactive with the TAT-positive serum samples. The combined rOmps sensitivity, specificity and accuracy were 215/232 (92.67%), 294/298 (98.66%) and 509/530 (96.04%), respectively. While these results are preliminary, the tests performed have very high potential in the serodiagnosis of brucellosis and likewise, the combined rOmps can be used for future vaccine production.