This is the first study demonstrating a protective effect of OMVs from a probiotic G- bacterium in a mouse model of allergic asthma. Here we describe the isolation, identification, biophysical characterisation, interaction with innate receptors and anti-allergic properties of E. coli O83 OMVs.
Although numerous epidemiological studies propose that continuous high exposure to environmental bacteria and endotoxins has a protective effect against allergic sensitisation and asthma, the exact mechanisms are not yet clear 51,52. Preclinical studies confirmed the beneficial effect of intranasal administration of farm dust 52,53 or farm dust microbes 54–56 and suggested that LPS is at least largely responsible for the beneficial effect 52,53,57. We have shown in our previous study that intranasally administered E. coli O83 reduced allergy in a TLR4-dependent manner, suggesting the role of LPS 17. Here we show that EcO83-OMVs, like their parent bacteria contain endotoxin and are recognised by TLR4. The effects of intranasal administration of LPS on pulmonary allergy are complex and to some extent controversial, with some studies showing a worsening, while others show a reduction in allergy 58,59. It is now understood that variables such as timing, dosage, chemical structure, and the resulting biological activity of the used LPS can affect the type of immune response that occurs 60.
In terms of timing, EcO83-OMVs were administered concomitantly with the allergen, at the time of allergic sensitization and challenge, using the same experimental protocol as we used for the application of live bacteria in our previous study 17. In this sense, Tulic et al. reported that Salmonella typhimurium LPS reduced pulmonary allergy when administered prior or up to 4 days after OVA sensitisation, but worsened it when administered more than 6 days after sensitisation 61. Furthermore, Bickert et al. have shown that LPS from E. coli 026:B6 reduced eosinophilia only when applied at the time of OVA challenge but not when administered before or after sensitisation 62. Whether prophylactic administration of vesicles prior to sensitisation and challenge has the potential to reduce allergy or whether the beneficial effect is long-lasting remains to be investigated.
It has been demonstrated that the dose of LPS delivered intranasally also determines the direction in which the immune system reacts. A high dose (100 µg) of LPS from a bacterial strain other than E. coli O83 reduced allergy to inhaled allergens in mice, but a low dose (0.1 µg) had the reverse effect 63. Here we show that the higher dose (1 µg) of EcO83-OMVs reduced allergy, whereas a lower dose (0.1 µg) did not worsen the disease severity but improved allergic inflammation in several parameters. Although LPS is a dominant OMV antigen, our results cannot be directly compared with those of Eisenbarth et al. also because they used LPS from other bacteria and it is known that LPS can vary greatly between different bacterial strains.
Flagella synthesis has been shown to play a key role in the budding of OMVs from E. coli64. Here we show that OMVs from E. coli O83 contain flagellin (FliC), a major structural protein of bacterial flagellum, and proteins required for anchoring the flagella in the membrane (flagellar hook-associated proteins FlgK, FlgL; hook protein FlgE; basal body rod proteins FlgB, FlgC, FlgE, FlgF; and L-ring protein FlgH). These proteins showed a significant fold enrichment in vesicles compared to the whole bacteria.. The cost of constructing flagella is high in bacteria such as E. coli, and the reason behind the release of these energy intensive molecules into the environment has yet to be investigated.
Antigen-presenting cells such as DCs recognise flagellin on their apical surface through TLR5, which activates NF-kB and MAPK, leading to the production of pro-inflammatory cytokines 65. In our studies, EcO83-OMVs, containing high levels of flagellin, induced the expression of pro-inflammatory cytokines such as IL-6, IL-12 and TNF-α in BMDCs, and the involvement of TLR5 in their recognition was confirmed in TLR5-transfected HEK293 cells. Vesicle-stimulated BMDCs also produce caspase-1-dependent IL-1β, suggesting activation of the NLR neuronal apoptosis inhibitory protein 5 or 6 (NAIP5/6), the sensor for intracellular flagellin, followed by assembly of the NLRC4 inflammasome 66. However, vesicle-induced IL-1β levels were several hundred-fold lower compared to levels induced by whole bacteria, suggesting that vesicles can be viewed as potent immunomodulators that lack some of the negative properties of the parent bacteria. On the other hand, vesicles but not whole bacteria were recognized by TLR5, implying that this receptor is important for vesicles recognition but less important for whole bacteria recognition.
In vivo, we have shown that intranasal treatment with vesicles reduces the number of eosinophils in the lungs compared to allergic controls. A similar effect was observed with intranasal application of recombinant flagellin 67,68. A recent study suggests that the binding of flagellin to surface receptors on eosinophils such as TLR5 may prevent eosinophil sensitisation 69. According to Luo et al., flagellin can reduce oxidative stress in eosinophils 68. We are currently conducting studies to elucidate the effect of EcO83-OMVs on oxidative stress in immune cells.
Not only immune cells but also airway structural cells can regulate innate immunity to allergens 70,71. We have shown that EcO83-OMVs are actively internalised by airway epithelial cells. Several pathways for internalisation of OMVs are known, such as macropinocytosis, endocytosis, membrane fusion or lipid raft formation 72. In a study by Canas et al., OMVs from probiotic and commensal E. coli strains entered epithelial cells via clathrin-mediated endocytosis and activated the intracellular receptor NOD1 26. The biomolecules of the bacterial cell wall, such as LPS, can influence the pathway of vesicular uptake and consequently the entry kinetics and efficiency 73. The specific pathway mediating the internalisation of EcO83-OMVs remains to be explored.
Recent studies have demonstrated the feasibility of using OMVs to deliver heterogeneous antigens. Eastwood et al. used an innovative expression system in E. coli based on a simple peptide tag that results in a high yield of functional proteins packaged into the vesicles 74. Another study used the “plug-and-play” approach to decorate S. typhimurium OMVs with the spike receptor-binding domain 75. This technology makes it possible to decorate the surface of OMVs with a variety of antigens or even multiple antigens. This could be of interest for the production of engineered OMVs decorated with allergens/peptides for specific immunotherapy. In our previous study, we constructed recombinant E. coli Nissle 1917 expressing the chimera with major birch and grass pollen allergenic protein/peptides and showed that its intranasal application prevented poly-sensitization in mice 76. Therefore, future studies in our laboratory will focus on developing a recombinant E. coli O83 to produce OMVs decorated with fluorescent markers or mono- and poly-allergens for specific immunotherapy.
In summary, we show that the probiotic E. coli O83 produces OMVs that exhibit potent immunomodulatory and anti-allergic properties. The ease of manufacture and the availability of technologies for genetically engineered vesicles, decorated with heterologous antigens in the lumen or on the surface, provide an extraordinarily versatile platform with great potential for therapeutic applications of bacterial vesicles in allergy research and clinic.