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Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

Khan Mirzaei M, Haileselassie Y, Navis M, Cooper C, Sverremark-Ekström E, Nilsson AS - Front Microbiol (2016)

Bottom Line: In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required.Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls.When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Stockholm, Sweden.

ABSTRACT
Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

No MeSH data available.


Related in: MedlinePlus

Cytokine release by peripheral blood mononuclear cells (PBMCs) following exposure to high concentrations (109 PFU/well) of four different purified bacteriophages. (A) IL-10 release. (B) IL-6 release. (C) TNF-α release. Data are the mean of two replicates taken from six donors ± SD. ∗∗P < 0.001; ∗P < 0.05 by Mann–Whitney. Cytokine induction by phages is compared to medium.
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Figure 2: Cytokine release by peripheral blood mononuclear cells (PBMCs) following exposure to high concentrations (109 PFU/well) of four different purified bacteriophages. (A) IL-10 release. (B) IL-6 release. (C) TNF-α release. Data are the mean of two replicates taken from six donors ± SD. ∗∗P < 0.001; ∗P < 0.05 by Mann–Whitney. Cytokine induction by phages is compared to medium.

Mentions: When assessed at the highest phage concentration (109 PFU/well) all samples were found to induce IL-6, IL-10, and TNF-α (Figure 2) at varying levels depending on the phage/cytokine combination. When compared to the medium (negative) control, IL-6 was significantly induced by all phages (P < 0.002 by Mann–Whitney) with the exception of SU32. Both IL-10 and TNF-α were significantly induced by SU57 and SU63 (P < 0.05 by Mann–Whitney) but not by SU10 or SU32 when compared to the negative control. When compared to the positive (LPS) control, all phages were significantly less immunostimulatory (P < 0.05 by Mann–Whitney) with the exception of SU63. TNF-α induction levels for SU57 were insignificant when compared to LPS. When comparing the ratio of TNF-α to IL-10, phage treated samples were higher than the medium control (P > 0.05 by Mann–Whitney). At lower phage concentrations (107 and 105 PFU/well) no significant induction of cytokines were observed (data not shown). IFN-γ was induced by all four phages but only in PBMCs obtained from two donors. The levels of IL-2, IL-4, and IL-17 were below the limit of detection for all conditions tested. In addition, no response to the purified bacterial debris when incubated with PBMC was observed (data not shown).


Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

Khan Mirzaei M, Haileselassie Y, Navis M, Cooper C, Sverremark-Ekström E, Nilsson AS - Front Microbiol (2016)

Cytokine release by peripheral blood mononuclear cells (PBMCs) following exposure to high concentrations (109 PFU/well) of four different purified bacteriophages. (A) IL-10 release. (B) IL-6 release. (C) TNF-α release. Data are the mean of two replicates taken from six donors ± SD. ∗∗P < 0.001; ∗P < 0.05 by Mann–Whitney. Cytokine induction by phages is compared to medium.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814447&req=5

Figure 2: Cytokine release by peripheral blood mononuclear cells (PBMCs) following exposure to high concentrations (109 PFU/well) of four different purified bacteriophages. (A) IL-10 release. (B) IL-6 release. (C) TNF-α release. Data are the mean of two replicates taken from six donors ± SD. ∗∗P < 0.001; ∗P < 0.05 by Mann–Whitney. Cytokine induction by phages is compared to medium.
Mentions: When assessed at the highest phage concentration (109 PFU/well) all samples were found to induce IL-6, IL-10, and TNF-α (Figure 2) at varying levels depending on the phage/cytokine combination. When compared to the medium (negative) control, IL-6 was significantly induced by all phages (P < 0.002 by Mann–Whitney) with the exception of SU32. Both IL-10 and TNF-α were significantly induced by SU57 and SU63 (P < 0.05 by Mann–Whitney) but not by SU10 or SU32 when compared to the negative control. When compared to the positive (LPS) control, all phages were significantly less immunostimulatory (P < 0.05 by Mann–Whitney) with the exception of SU63. TNF-α induction levels for SU57 were insignificant when compared to LPS. When comparing the ratio of TNF-α to IL-10, phage treated samples were higher than the medium control (P > 0.05 by Mann–Whitney). At lower phage concentrations (107 and 105 PFU/well) no significant induction of cytokines were observed (data not shown). IFN-γ was induced by all four phages but only in PBMCs obtained from two donors. The levels of IL-2, IL-4, and IL-17 were below the limit of detection for all conditions tested. In addition, no response to the purified bacterial debris when incubated with PBMC was observed (data not shown).

Bottom Line: In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required.Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls.When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Stockholm, Sweden.

ABSTRACT
Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

No MeSH data available.


Related in: MedlinePlus