Studies on the host immune responses induced by extracellular vesicles derived from Gram-negative bacteria

Abstract

Extracellular vesicles (EVs) are spherical bi-layered phospholipids ranging in size from 20-200 nm in diameter that are produced from bacteria, archea and eukaryotic cells. EVs derived from Gram-negative bacteria can induce various immune responses to the host. These inductions of immune responses may be harmful or beneficial to the host. Recent evidence indicates that Gram-negative bacteria?derived extracellular vesicles (EVs) in indoor dust can evoke neutrophilic pulmonary inflammation, which is a key pathology of chronic obstructive pulmonary disease (COPD). Escherichia coli is a ubiquitous bacterium present in indoor dust and secretes nanometer-sized vesicles into the extracellular milieu. Firstly, I evaluated the role of E. coli?derived EVs on the development of COPD, such as emphysema. E. coli EVs were prepared by sequential ultrafiltration and ultracentrifugation. COPD phenotypes and immune responses were evaluated in C57BL/6 wild-type (WT), IFN-g?deficient, or IL-17A?deficient mice after airway exposure to E. coli EVs. The present study showed that indoor dust from a bed mattress harbors E. coli EVs. Airway exposure to E. coli EVs increased the production of pro-inflammatory cytokines, such as TNF-a and IL-6. In addition, the repeated inhalation of E. coli EVs for 4 weeks induced neutrophilic inflammation and emphysema, which are associated with enhanced elastase activity. Emphysema and elastase activity enhanced by E. coli EVs were reversed by the absence of IFN-g or IL-17A genes. In addition, during the early period, lung inflammation is dependent on IL-17A and TNF-a, but not on IFN-g, and also on TLR4. Moreover, the production of IFN-g is eliminated by the absence of IL-17A, whereas IL-17A production is not abolished by IFN-g absence. Taken together, the present data suggest that E. coli?derived EVs induce IL-17A?dependent neutrophilic inflammation and thereby emphysema, possibly via upregulation of elastase activity. The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived EVs are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, I evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the up-regulation of co-stimulatory molecule expression and pro-inflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. 3 times vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections. Recent vaccine R&D has brought attention to the use of outer membrane vesicles (OMVs), extracellular vesicles (EVs) secreted from gram-negative bacteria, as a new vaccine vehicle. However, OMVs possess toxicity issue, because OMVs harbor bacterial toxins, including lipopolysaccharide (LPS), and also have manufacturability issue. To avoid toxicity of OMV-based vaccine, I used bacterial protoplast, which is manufactured by the removal of outer membrane and cell wall components. And then, to overcome the productivity issue of native EVs, vesicles were manufactured artificially from bacterial protoplast, and called by protoplast-derived bionanosome (P-BNS), as a novel antigen delivery system. P-BNS vaccine candidates against K. pneumoniae infections were made by loading with target antigens into E. coli. In vitro and in vivo innate and adaptive immunogenicity of antigen-loaded P-BNS was evaluated by its ability to induce pro-inflammatory mediators and induce antigen-specific adaptive immunity. To examine the efficacy of P-BNS vaccination in the mouse, P-BNS vaccine candidates were actively immunized in mice challenged with K. pneumoniae. The current study showed that antigen-loaded PNS vaccine candidates induced antigen-specific antibody and T cell responses. However, the lethality induced by K. pneumoniae infection effectively prevented by only K. pneumoniae-specific OMPA-loaded P-BNS effectively protected but not by other antigen-loaded P-BNS. To sum up, these findings indicate active immunization with P-BNS loaded with bacterial surface OMPA antigen conferred effective protection against lethality induced by K. pneumoniae infection.