Background: Lactobacillus reuteri strains are widely used as probiotics to prevent and treat inflammatory bowel disease by modulating the host’s immune system. However, the underlying mechanisms by which they communicate with the host have not been clearly understood. Bacterial extracellular vesicles (EVs) have been considered as important mediators of host-pathogen interactions, but their potential role in commensals-host crosstalk has not been widely studied. Here, we investigated the regulatory actions of EVs produced by L. reuteri BBC3, a gut-associated commensal bacterium of Black-Bone chicken, in the development of lipopolysaccharide (LPS)-induced intestinal inflammation in a chicken model using both in vivo and in vitro experiments. Results: L. reuteri BBC3 produced nano-scale membrane vesicles with the size range of 60–250 nm. Biochemical and proteomic analyses showed that L. reuteri BBC3-derived EVs (LrEVs) carried DNA, RNA and several bioactive proteins previously described as mediators of other probiotics’ beneficial effects such as glucosyltransferase, serine protease and elongation factor Tu. In vivo broiler experiments showed that administration of LrEVs exerted similar effects as L. reuteri BBC3 in attenuating LPS-induced inflammation by improving growth performance, reducing mortality and decreasing intestinal injury. LrEVs suppressed the LPS-induced expression of pro-inflammatory genes (TNF-α, IL-1β, IL-6, IL-17 and IL-8), and improved the expression of anti-inflammatory genes (IL-10 and TGF-β) in the jejunum. LrEVs could be internalized by chicken macrophages. In vitro pretreatment with LrEVs reduced the gene expression of TNF-α, IL-1β and IL-6 by suppressing the NF-κB activity, and enhanced the gene expression of IL-10 and TGF-β in LPS-activated chicken macrophages. Additionally, LrEVs could inhibit Th1- and Th17-mediated inflammatory responses and enhance the immunoregulatory cells-mediated immunosuppression in splenic lymphocytes of LPS-challenged chickens through the activation of macrophages. Finally, we revealed that the reduced content of both vesicular proteins and nucleic acids attenuated the suppression of LrEVs on LPS-induced inflammatory responses in ex vivo experiments, suggesting that they are essential for the LrEVs-mediated immunoregulation. Conclusions: We revealed that LrEVs participated in maintaining intestinal immune homeostasis against LPS-induced inflammatory responses in a chicken model. Our findings provide mechanistic insight into how commensal and probiotic Lactobacillus species modulate the host’s immune system in pathogens-induced inflammation.

Background: ProbioticEscherichia coliNissle 1917 (EcN) has been widely studied for the treatment of intestinal inflammatory diseases and infectious diarrhea, but the mechanisms by which they communicate with the host are not well-known. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in the host, which play a very important role in mediating bacteria-host communication. Here, we aimed to investigate whether EcN-derived OMVs (EcN_OMVs) could mediate immune regulation in macrophages.Results: In this study, after the characterization of EcN_OMVs using electron microscopy, nanoparticle tracking and proteomic analyses, we demonstrated by confocal fluorescence microscopy that EcN_OMVs could be internalized by RAW 264.7 macrophages. Stimulation with EcN_OMVs at appropriate concentrations promoted proliferation, immune-related enzymatic activities and phagocytic functions of RAW264.7 cells. Moreover, EcN_OMVs induced more anti-inflammatory responses (IL-10) than pro-inflammatory responses (IL-6 and TNF-α) in vitro, and also modulated the production of Th1-polarizing cytokine (IL-12) and Th2-polarizing cytokine (IL-4). Treatments with EcN_OMVs effectively improved the antibacterial activity of RAW 264.7 macrophages.Conclusions: These findings indicated that EcN_OMVs could modulate the functions of the host immune cells, which will enrich the existing body of knowledge of EVs as an important mechanism for the communication of probiotics with their hosts.

Background: The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. Results: In this study, OMVs produced by three APEC strains were isolated, purified and prepared into MOMVs by mixing these three OMVs. By using SDS-PAGE and LC–MS/MS, 159 proteins were identified in MOMVs and the subcellular location and biological functions of 20 most abundant proteins were analyzed. The immunogenicity of MOMVs was evaluated, and the results showed that MOMVs could elicit innate immune responses, including internalization by chicken macrophage and production of immunomodulatory cytokines. Vaccination with MOMVs induced specific broad-spectrum antibodies as well as Th1 and Th17 immune responses. The animal experiment has confirmed that immunization with an appropriate dose of MOMVs could not cause any adverse effect and was able to reduce bacteria loads and pro-inflammatory cytokines production, thus providing effective cross-protection against lethal infections induced by multi-serogroup APEC strains in chickens. Further experiments indicated that, although vesicular proteins were able to induce stronger protective efficiency than lipopolysaccharide, both vesicular proteins and lipopolysaccharide are crucial in MOMVs-mediated protection. Conclusions: The multi-serogroup nanovesicles produced by APEC strains will open up a new way for the development of next generation vaccines with low toxicity and broad protection in the treatment and control of APEC infection.

B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter Pglv. The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the Pglv promoter system and enhance its expression strength.

Avian pathogenic Escherichia coli (APEC) infection in poultry causes enormous economic losses and public health risks. Bacterial outer membrane vesicles (OMVs) and nano-sized proteolipids enriched with various immunogenic molecules have gained extensive interest as novel nanovaccines against bacterial infections. In this study, after the preparation of APEC O2-derived OMVs (APEC_OMVs) using the ultracentrifugation method and characterization of them using electron microscopy and nanoparticle tracking analyses, we examined the safety and vaccination effect of APEC_OMVs in broiler chicks and investigated the underlying immunological mechanism of protection. The results showed that APEC_OMVs had membrane-enclosed structures with an average diameter of 89 nm. Vaccination with 50 μg of APEC_OMVs had no side effects and efficiently protected chicks against homologous infection. APEC_OMVs could be effectively taken up by chicken macrophages and activated innate immune responses in macrophages in vitro. APEC_OMV vaccination significantly improved activities of serum non-specific immune factors, enhanced the specific antibody response and promoted the proliferation of splenic and peripheral blood lymphocytes in response to mitogen. Furthermore, APEC_OMVs also elicited a predominantly IFN-γ-mediated Th1 response in splenic lymphocytes. Our data revealed the involvement of both non-specific immune responses and specific antibody and cytokine responses in the APEC_OMV-mediated protection, providing broader knowledge for the development of multivalent APEC_OMV-based nanovaccine with high safety and efficacy in the future.