Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages.

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1ß, IL-6, IL-10, and TGF-ß1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

Enhancing effect of chitosan nanoparticles on the immune efficacy of Bordetella bronchiseptica outer membrane vesicles.

The outer membrane vesicle (OMV) of bacteria is a bilayer membrane vesicle with a diameter of about 10-300 nm that is secreted during the growth of Gram-negative bacteria. OMV is considered as a high-quality vaccine candidate antigen because of its natural immunogenicity and non-replicability. Although the excellent antigenicity of OMV has been widely confirmed, its instability and heterogeneity greatly affect its immune effect. Many studies have demonstrated that in combination with nanoparticles can enhance the stability of OMV. In this study, OMVs were used to coat chitosan nanoparticles (CNPs) and obtain a stable OMV vaccine. The characteristics, including morphology, hydrodynamic size, and zeta potential were evaluated. The immune protection of CNP-OMV and anti-infection efficacy were examined and compared in vivo and in vitro. The results showed that the CNP-OMV were homogenous with a size of 139 nm and a stable core-shell structure. And CNP-OMV could significantly increase the cell proliferation, phagocytosis and TNF-α, IL-6 and IL-10 secretion of RAW264.7 in vitro. In vivo, CNP-OMV could significantly increase the levels of anti-Bb and OMV IgG antibodies. Levels of blood lymphocyte, and Th1 (IFN-γ, IL-12), Th2 (IL-4, IL-5), and Th17 (IL-17, TNF-α) type cytokines in the serum were all significantly increased. At the same time, CNP-OMV could significantly reduce the bacterial invading the lungs of challenged rabbits. And CNP-OMV could largely protect the lungs from injury. The above results showed that CNP-OMV had a good immune efficacy and could resist the infection of Bordetella bronchiseptica. This study provided a scientific basis for the development of novel effective and safe vaccine against Bordetella bronchiseptica, and also provided a new idea for the development of new bacterial vaccine.

Adjuvant Efficacy of the ECMS-Oil on Immune Responses against Bordetella bronchiseptica in Mice through the TLR2/MyD88/NF-κB Pathway.

Bordetella infection can be efficiently prevented through vaccination. The current study investigated the effects of an extract of Cochinchina momordica seed (ECMS) combined with oil on the immune responses to the inactivated Bordetella vaccine in mice. Serum IgG and IgG1 level was significantly increased in ECMS-oil group compared to any other group (P < 0.05) 2 weeks after immunization, while groups ECMS200 µg/400 µg-oil had a markedly higher level of serum IgG2b and IgG3 than any other groups (P < 0.05). Moreover, lipopolysaccharide/ConA-stimulated proliferation of splenocytes was significantly enhanced in ECMS 400 µg-oil immunized mice in comparison with mice in any other group (P < 0.05). RT-PCR assay revealed that while ECMS800 µg-oil group had significantly higher levels of serum IL-4, IL-10, Toll-like receptor (TLR)2, and IL-1 beta than any other group (P < 0.05), the levels of serum IL-2, IL-4, and IL-10 were markedly increased in ECMS 400 µg-oil group as compared to any other groups (P < 0.05). Blood analysis showed that ECMS800 µg-oil and oil groups had a significantly higher number of immunocytes than any other groups (P < 0.05). There were significant differences in the number of IgG+, IgG2b+, and IgA+ cells in the lung between ECMS800 µg-oil group and any other groups (P < 0.05). Western blot analysis demonstrated that stimulation with ECMS 25 µg/mL or 50 ng/mL led to a significant increase in the expression of TLR2, MyD88, and NF-κB in Raw264.7 cells (P < 0.05). Compared with any other group, the expression of MyD88 was markedly increased in the cells stimulated with ECMS 50 ng/mL, as indicated by the RT-PCR analysis (P < 0.05). Overall, we observed that ECMS-oil efficiently enhanced the humoral or cellular immune responses against Bordetella and suggested that the mechanism of adjuvant activity of ECMS-oil might involve TLR2/MyD88/NF-κB signaling pathway.

Safety and Efficacy of the Bordetella bronchiseptica Vaccine Combined with a Vegetable Oil Adjuvant and Multi-Omics Analysis of Its Potential Role in the Protective Response of Rabbits.

Infectious respiratory diseases caused by Bordetella bronchiseptica (Bb) are seriously endangering the development of the rabbit industry in China. Unfortunately, no licensed vaccines are available for this pathogen. The present study was designed to determine whether the inactivated Bb antigen formulated with vegetable oil adjuvant (named E515) which contains soybean oil, vitamin E, and ginseng saponins, functions as a safe and effective vaccine (E515-Bb) against Bb infection in rabbits. Based on local and systemic reactions, both the E515 adjuvant alone and the E515-Bb vaccine exhibited good safety in rabbits. Immune response analysis implies that rabbits immunized with the E515-Bb vaccine produced significantly higher, earlier, and longer-lasting specific antibody responses and activated Th1/Th2/Th17 cell responses than those immunized with the aluminum hydroxide (Alum)-adjuvanted Bb vaccine (Alum-Bb) or Bb antigen alone. Moreover, the E515-Bb vaccine effectively protected rabbits from Bb infection. Additionally, integrated multi-omics analysis revealed that the immunoprotective effect of the E515-Bb vaccine was achieved through upregulation of the complement and coagulation cascades and cell adhesion molecule (CAM) pathways, and the downregulation of the P53 pathway. Overall, these results indicate that the E515-Bb vaccine is safe, elicits an efficient immune response and provides good protection against Bb infection in rabbits. Thus, the E515-adjuvanted Bb vaccine can be considered a promising candidate vaccine for preventing Bb infection.

Optimum Preparation Method for Self-Assembled PEGylation Nano-Adjuvant Based on Rehmannia glutinosa Polysaccharide and Its Immunological Effect on Macrophages.

BACKGROUND: Rehmannia glutinosa polysaccharide is the main reason that contributes to the immunological function of R. glutinosa. Due to its disadvantages in clinical use, here we designed the PEGylation nano-RGP (pRL) to improve the drug-targeting effect and the immunological function. Our present work aims to establish the optimum condition of preparing the pRL and to investigate its immunological function on macrophages. METHODS: pRL was prepared by thin film hydration method combined with ultra-sonication technique. And its preparation conditions were optimized with response surface methodology. Also, the lyophilization method was optimized. The characteristics of the pRL were evaluated, including particle size, drug loading, encapsulation efficiency and morphology. The immunological function of pRL on macrophage was investigated through CCK-8 test, ELISA and flow cytometry. RESULTS: The lipid-to-cholesterol molar ratio of 8:1, the addition of DSPE-PEG2000 of 9% and the lipid-to-drug ratio of 5.4:1 were the optimum preparation technology for pRL. The encapsulation efficiency (EE) of pRL under this preparation technology was 95.81±1.58%, with a particle size of 31.98 ± 2.6 nm. The lactose-to-lipid ratio (2:1) was the optimal lyophilization method. pRL promoted macrophage proliferation, which is significantly better than that of nano-RGP without PEGylation (RL). pRL-stimulated RAW264.7 cells showed a high secretion of pro-inflammatory cytokines, which is the characteristic indicator of M1 polarization. Enhanced cellular uptake through macropinocytosis-dependent and caveolae-mediated endocytosis was observed in pRL-treated RAW264.7 cells. CONCLUSION: Our study concluded that PEGylation effectively overcame the poor targeting effect of Rehmannia glutinosa polysaccharide (RGP) and significantly improved the immunological profile of its nano-formulation, which suggested that pRL could serve as an immune adjuvant in clinical application.

Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen.

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

Slc7a11 Modulated by POU2F1 is Involved in Pigmentation in Rabbit.

Solute carrier family 7 member 11 (Slc7a11) is a cystine/glutamate xCT transporter that controls the production of pheomelanin pigment to change fur and skin color in animals. Previous studies have found that skin expression levels of Slc7a11 varied significantly with fur color in Rex rabbits. However, the molecular regulation mechanism of Slc7a11 in pigmentation is unknown. Here, rabbit melanocytes were first isolated and identified. The distribution and expression pattern of Slc7a11 was confirmed in skin from rabbits with different fur colors. Slc7a11 affected the expression of pigmentation related genes and thus affected melanogenesis. Meanwhile, Slc7a11 decreased melanocyte apoptosis, but inhibition of Slc7a11 enhanced apoptosis. Furthermore, the POU2F1 protein was found to bind to the -713 to -703 bp region of Slc7a11 promoter to inhibit its activity in a dual-luciferase reporter and site-directed mutagenesis assay. This study reveals the function of the Slc7a11 in melanogenesis and provides in-depth analysis of the mechanism of fur pigmentation.

Greater efficacy of the ECMS-oil adjuvant over other formulations on immune responses against Bordetella bronchiseptica in rabbits and the underlying mechanism.

In this study, the adjuvant effects of the extract of Cochinchina momordica seed ECMS+oil, oil alone, ECMS alone, conventional alum adjuvant on inactivated Bordetella bronchiseptica (Bb) vaccine or control using antigen alone without adjuvant were evaluated along with the underlying mechanism. The results in experiment A demonstrated that antibody levels in Bb whole cell protein in the ECMS800µg+oil group were significantly higher than in the other adjuvant groups (p<0.05) on day 21. The agglutination antibody titer was also higher than the other groups (p<0.05) on day 37. The ECMS800µg+oil group improved cellular immune responses compared to other adjuvant groups, including control using antigen alone without adjuvant and the PBS group (p<0.05). After Bb challenge, the ECMS800µg+oil group showed the highest protection rate, which was significantly higher than ECMS alone or control using antigen alone without adjuvant and the PBS group (p<0.05 and p<0.01). IgA cells in the ECMS800µg+oil group differed significantly from the IgA cells of other groups in the lungs (p<0.01). The results of cell recruitment showed that the number of lymphocytes in the ECMS400µg+oil were higher than the number of cells for other groups except the ECMS(100µg/800µg)+oil groups (p<0.05). Intermediate cells in the ECMS(100µg/400µg)+oil groups were higher than the number of cells for other groups, including the control using antigen alone group (p<0.05). Neutrophils in the ECMS(100µg/400µg/800µg)+oil groups were significantly higher than the ECMS 800µg and control using antigen alone groups (p<0.05). White blood cells in the ECMS100µg+oil group were significantly higher than the oil, ECMS800µg and control using antigen alone groups (p<0.05). IL-2 expression in the ECMS800µg+oil group was significantly higher than other groups, except for the ECMS400µg+oil group (p<0.05). IL-4 expression in the ECMS800µg+oil group was significantly higher than other groups (p<0.05). GATA3 in the ECMS800µg+oil groups was significantly higher than the oil, ECMS800µg and control using antigen alone group (p<0.05). ECMS-oil adjuvant mixture could most effectively protect B. bronchiseptica immunized rabbits and, therefore, could be an alternative way of improving B. bronchiseptica vaccination in rabbits.