The Impact of Escherichia coli Probiotic Strain O83:K24:H31 on the Maturation of Dendritic Cells and Immunoregulatory Functions In Vitro and In Vivo.

Early postnatal events are important for the development of the neonatal immune system. Harboring the pioneering microorganisms forming the microbiota of the neonatal gastrointestinal tract is important for priming the immune system, as well as inducing appropriate tolerance to the relatively innocuous environmental antigens and compounds of normal healthy microbiota. Early postnatal supplementation of suitable, safe probiotics could accelerate this process. In the current study, the immunomodulatory capacity of the probiotic strain of Escherichia coli O83:K24:H31 (EcO83) was characterized in vitro and in vivo. We compared the capacity of EcO83 with and without hemolytic activity on selected immune characteristics in vitro as determined by flow cytometry and quantitative real-time PCR. Both strains with and without hemolytic activity exerted comparable capacity on the maturation of dendritic cells while preserving the induction of interleukin 10 (Il10) expression in dendritic cells and T cells cocultured with EcO83 primed dendritic cells. Early postnatal supplementation with EcO83 led to massive but transient colonization of the neonatal gastrointestinal tract, as detected by in vivo bioimaging. Early postnatal EcO83 administration promoted gut barrier function by increasing the expression of claudin and occludin and the expression of Il10. Early postnatal EcO83 application promotes maturation of the neonatal immune system and promotes immunoregulatory and gut barrier functions.

Effect of early postnatal supplementation of newborns with probiotic strain E. coli O83:K24:H31 on allergy incidence, dendritic cells, and microbiota.

Introduction: Probiotic administration seems to be a rational approach to promote maturation of the neonatal immune system. Mutual interaction of the microbiota with the host immune system is critical for the setting of appropriate immune responses including a tolerogenic one and thevmaintenance of homeostasis. On the other hand, our knowledge on the modes of actions of probiotics is still scarce. Methods: In our study, probiotic strain Escherichia coli O83:K24:H31 (EcO83) was administered to neonates of allergic mothers (AMs; neonates with increased risk for allergy development) within 48 h after the delivery, and the impact of this early postnatal supplementation on allergy incidence and selected immune markers has been analyzed 10 years after the primary EcO83 administration. Results: We have observed decreased allergy incidence in 10-year-old children supplemented with EcO83 (13 of 52 children were allergic) in comparison with non-supplemented children of AMs (16 of 42 children were allergic). The early postnatal EcO83 supplementation appeared to limit the allergy in the high-risk group (children of AMs) compared to that in the low-risk group (children of healthy mothers). Dendritic cells (DCs) in the peripheral blood of EcO83-supplemented children do not differ significantly in cell surface presence of CD83. The immunomodulatory capacity of EcO83 on DCs was tested in vitro as well. Both directly isolated myeloid and in vitro monocyte-derived DCs from cord blood increased CD83 expression together with interleukin (IL)-10 secretion after EcO83 stimulation. The effect of early postnatal EcO83 supplementation on the microbiota composition of 10-year-old children was characterized by next-generation sequencing, and we have not observed significant changes in the microbiota composition of EcO83-supplemented and non-supplemented children at the age of 10 years. Conclusions: Early postnatal EcO83 supplementation appears to lower allergy incidence in children of AMs. It seems that the beneficial effect of EcO83 is mediated via modulation of DC functional capacities without impacting the microbiota composition. Larger-scale studies will be necessary to confirm these preliminary findings.

In Vitro Characterization of Gut Microbiota-Derived Commensal Strains: Selection of Parabacteroides distasonis Strains Alleviating TNBS-Induced Colitis in Mice.

Alterations in the gut microbiota composition and diversity seem to play a role in the development of chronic diseases, including inflammatory bowel disease (IBD), leading to gut barrier disruption and induction of proinflammatory immune responses. This opens the door for the use of novel health-promoting bacteria. We selected five Parabacteroides distasonis strains isolated from human adult and neonates gut microbiota. We evaluated in vitro their immunomodulation capacities and their ability to reinforce the gut barrier and characterized in vivo their protective effects in an acute murine model of colitis. The in vitro beneficial activities were highly strain dependent: two strains exhibited a potent anti-inflammatory potential and restored the gut barrier while a third strain reinstated the epithelial barrier. While their survival to in vitro gastric conditions was variable, the levels of P. distasonis DNA were higher in the stools of bacteria-treated animals. The strains that were positively scored in vitro displayed a strong ability to rescue mice from colitis. We further showed that two strains primed dendritic cells to induce regulatory T lymphocytes from naïve CD4+ T cells. This study provides better insights on the functionality of commensal bacteria and crucial clues to design live biotherapeutics able to target inflammatory chronic diseases such as IBD.

Inhibition of Pro-Inflammatory Cytokines by Metabolites of Streptomycetes-A Potential Alternative to Current Anti-Inflammatory Drugs?

Current treatment of chronic diseases includes, among others, application of cytokines, monoclonal antibodies, cellular therapies, and immunostimulants. As all the underlying mechanisms of a particular diseases are not always fully clarified, treatment can be inefficient and associated with various, sometimes serious, side effects. Small secondary metabolites produced by various microbes represent an attractive alternative as future anti-inflammatory drug leads. Compared to current drugs, they are cheaper, can often be administered orally, but still can keep a high target-specificity. Some compounds produced by actinomycetes or fungi have already been used as immunomodulators-tacrolimus, sirolimus, and cyclosporine. This work documents strong anti-inflammatory features of another secondary metabolite of streptomycetes-manumycin-type polyketides. We compared the effect of four related compounds: manumycin A, manumycin B, asukamycin, and colabomycin E on activation and survival of human monocyte/macrophage cell line THP-1. The anti-cancer effect of manucycine A has been demonstrated; the immunomodulatory capacities of manumycin A are obvious when using micromolar concentrations. The application of all four compounds in 0.25-5 µM concentrations leads to efficient, concentration-dependent inhibition of IL-1ß and TNF expression in THP-1 upon LPS stimulation, while the three latter compounds show a significantly lower pro-apoptotic effect than manumycin A. We have demonstrated the anti-inflammatory capacity of selected manumycin-type polyketides.

The peripheral immune response of mice infected with a neuropathogenic schistosome.

Trichobilharzia regenti (Schistosomatidae) percutaneously infects birds and mammals and invades their central nervous system (CNS). Here, we characterized the peripheral immune response of infected mice and showed how it was influenced by the parasite-induced inflammation in the skin and the CNS. As revealed by flow cytometry, T cells expanded in the spleen and the CNS-draining lymph nodes 7-14 days post-infection. Both T-bet+ and GATA-3+ T cells were markedly elevated suggesting a mixed type 1/2 immune response. However, it dropped after 7 dpi most likely being unaffected by the neuroinflammation. Splenocytes from infected mice produced a high amount of IFN-γ and, to a lesser extent, IL-10, IL-4 and IL-17 after in vitro stimulation by cercarial homogenate. Nevertheless, it had only a limited capacity to alter the maturation status of bone marrow-derived dendritic cells (BMDCs), contrary to the recombinant T. regenti cathepsin B2, which also strongly augmented expression of Ccl5, Cxcl10, Il12a, Il33 and Il10 by BMDCs. Taken together, mice infected with T. regenti developed the mixed type 1/2 immune response, which was driven by the early skin inflammation rather than the late neuroinflammation. Parasite peptidases might play an active role in triggering the host immune response.

Evaluation of the Immunogenicity of a Vascular Graft Covered with Collagen Derived from the European Carp (Cyprinus carpio) and Bovine Collagen.

AIM: To assess the systemic and local immunological response to subcutaneous implants of a vascular graft covered with collagen extracted from the European carp (freshwater fish) or with collagen of bovine origin. METHODS: Pieces of a vascular graft covered by pure bovine (Bos taurus, BOV, n=14) or carp (Cyprinus carpio, CYP, n=14) collagen 5 mm in size were implanted subcutaneously in the dorsum of a Balb/cOla mice. A sham operation group of 12 animals served as the control. At 7 and 14 days after the operation, one-half of each group was terminated and blood for serum, spleen, and implant with surrounding tissue were collected. Mean cytokine (TNF-α, IL-10, IL-4, IL-1ß, IL-13, and IFN-γ) levels in serum were determined using ELISA. Spleen cell cultures were used for in vitro testing of lymphocyte proliferation and cytokine secretion. Local expressions of IL-6, IL-10, TNF-α, TGF-ß, CCL-2, and CCL-3 were determined using PCR. RESULTS: We found no significant difference among control, BOV, and CYP groups in mean cytokine serum levels at seven days. At day 14, the BOV group had higher levels of TNF-α (P=.018) and both the BOV and CYP groups had lower levels of IL-4 (P=.011 and P=.047, respectively) compared with the control group. Both tested implants showed only a minimal effect on the production of selected cytokines. Cell proliferation in the CYP group stimulated by CYP gel at 14 days was significantly lower than by BOV gel in BOV group (P=.0031) or by CYP gel in the control group (P=.041). The difference between the groups in the local RNA expression of all the tested mediators both at 7 and at 14 days was not significant apart from a lower level of TNF-α in the BOV group compared to CYP at 14 days (P=.013). CONCLUSIONS: Implants covered with carp collagen induce an immunological response that is comparable to that of bovine collagen covered implants in a mouse model.

Decreased allergy incidence in children supplemented with E. coli O83:K24:H31 and its possible modes of action.

The growing knowledge of the key role of microbiota in the maturation of neonatal immune system suggests that manipulation of microbiota could be exploited in hampering allergy development. In this study, Escherichia coli O83:K24:H31 (EcO83) was administered to newborns that were followed prospectively. Several immunological characteristics (cytokines, specific IgE, total T regulatory cells (Treg) and subpopulation of natural Treg (nTreg) and induced Treg (iTreg)) were tested in peripheral blood of 8-year-old children. Incidence of allergic disease was decreased in EcO83 supplemented children and significantly elevated levels of IL-10 and IFN-É£ were detected in serum of EcO83 supplemented children. Probiotic supplementation did not influence the numbers of the total Treg population but their functional capacity (intracellular expression of IL-10) was significantly increased in children supplemented with EcO83 in comparison to non-supplemented children. Morover, decreased proportion of iTreg was present in peripheral blood of non-supplemented in comparison to EcO83 supplemented children. Finally, stimulation of cord blood cells with EcO83 promoted both gene expression and secretion of IL-10 and IFN-É£ suggesting that beneficial effect of EcO83 in prevention of allergy development could be mediated by promotion of regulatory responses (by IL-10) and Th1 immune response (by IFN-É£).

Different capacity of in vitro generated myeloid dendritic cells of newborns of healthy and allergic mothers to respond to probiotic strain E. coli O83:K24:H31.

Allergic diseases belong to one of the most common diseases with steadily increasing incidence even among young children. There is an urgent need to identify a prognostic marker pointing to increased risk of allergy development enabling early preventive measures introduction. It has been shown that administration of selected probiotic strains or mixtures could prevent allergy development. In our study, we have tested the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to promote dendritic cell (DC) maturation and polarisation of immune responses. Increased presence of activation marker CD83 was observed on DC stimulated by E. coli O83 and DC of newborns of allergic mothers have significantly more increased cell surface presence of CD83 in comparison to children of healthy mothers. Increased gene expression and secretion of IL-10 was detected in DC stimulated with E. coli O83 being higher in DC of newborns of healthy mothers in comparison to allergic ones. Generally, increased presence of intracellular cytokines (IL-4, IL-13, IFN-gamma, IL-17A, IL-22, IL-10) was detected in CD4+ T cells cocultured with DC of children of allergic mothers in comparison to healthy ones. E. coli O83 primed DC significantly increased IL-10 and IL-17A in CD4 T cells of newborns of healthy mothers in comparison to the levels detected in CD4 T cells cocultured with control non-stimulated DC. We can conclude E. coli O83 induces dendritic cell maturation and IL-10 production in DC. Newborns of allergic mothers have generally increased reactivity of both DC and CD4 T cells which together with decreased capacity of DC of newborns of allergic mothers to produce IL-10 could support inappropriate immune responses development after allergen encounter.