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Mammalian Host-Versus-Phage immune response determines phage fate in vivo.

Hodyra-Stefaniak K, Miernikiewicz P, Drapała J, Drab M, Jończyk-Matysiak E, Lecion D, Kaźmierczak Z, Beta W, Majewska J, Harhala M, Bubak B, Kłopot A, Górski A, Dąbrowska K - Sci Rep (2015)

Bottom Line: Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy.We accommodated the experimental data into a mathematical model.We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations.

View Article: PubMed Central - PubMed

Affiliation: Bacteriophage Laboratory, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland.

ABSTRACT
Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy.

No MeSH data available.


Related in: MedlinePlus

Visualization of bacteriophage phagocytosis in spleen macrophages up-taking the model phage.The particles of GFP-expressing bacteriophage are visible in discrete locations of the macrophage – as clusters of viral particles. Spectral unmixing confocal microscopy of GFP variants (A,B) powered by super-resolution microscopy (structural illumination mode, (B–D)) of macrophages exposed to phages. A combination of the two complementary imaging modes provides specific information about the presence of native and partially degraded GFP-labeled phages, allows them to be distinguished from the auto-fluorescence background and provides a super-resolved image reconstruction of the phage particle localization pattern. SEM imaging of the phage alone and during phagocytosis by macrophages (F–I). (A) Lambda scan-generated spectra of the enhanced-GFP native form (green curve), the partially degraded form of GFP (red-shifted GFP spectrum, here the red curve), and the auto-fluorescence (magenta curve). (B) Typical morphology of the cell loaded with GFP-labeled phages, spectrally unmixed image showing the native-label particles (green), particles with partially degraded GFP label (red) and auto-fluorescence (magenta). Scale bar = 6 μm (C) Super-resolution imaging of a representative macrophage with the phage particles. Several clusters of particles visible (GFP-labeled phage). Scale bar = 6 μm. (D) Inset showing representative cluster of phages within a macrophage. Scale bar = 1 μm. (E) Dimensions of a representative cluster (yellow set) and the estimated measurements of a single phage (red set). The values of particle size approximate the expected dimensions of a phage virus labeled by multiple GFP molecules. Scale bar = 0.6 μm. (F) Set of representative macrophages during the uptake of phage particles, SEM scanned at low beam accelerating voltages with SE2 detection at 3 kV acceleration voltage. Scale-bar = 5 μm. (G) A representative macrophage during the uptake of phage particles, in lens SE detection at 3 kV acceleration voltage. Scale bar = 1 μm. (H) Phage particles on silicon, without macrophages, in-lens SE detection (3 kV). Scale bar = 100 nm. (I) Inset showing the uptake of phages into a macrophage, a magnified view of the inset region (red box) in (G) in lens SE detection (3 kV). Scale bar = 100 nm.
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f2: Visualization of bacteriophage phagocytosis in spleen macrophages up-taking the model phage.The particles of GFP-expressing bacteriophage are visible in discrete locations of the macrophage – as clusters of viral particles. Spectral unmixing confocal microscopy of GFP variants (A,B) powered by super-resolution microscopy (structural illumination mode, (B–D)) of macrophages exposed to phages. A combination of the two complementary imaging modes provides specific information about the presence of native and partially degraded GFP-labeled phages, allows them to be distinguished from the auto-fluorescence background and provides a super-resolved image reconstruction of the phage particle localization pattern. SEM imaging of the phage alone and during phagocytosis by macrophages (F–I). (A) Lambda scan-generated spectra of the enhanced-GFP native form (green curve), the partially degraded form of GFP (red-shifted GFP spectrum, here the red curve), and the auto-fluorescence (magenta curve). (B) Typical morphology of the cell loaded with GFP-labeled phages, spectrally unmixed image showing the native-label particles (green), particles with partially degraded GFP label (red) and auto-fluorescence (magenta). Scale bar = 6 μm (C) Super-resolution imaging of a representative macrophage with the phage particles. Several clusters of particles visible (GFP-labeled phage). Scale bar = 6 μm. (D) Inset showing representative cluster of phages within a macrophage. Scale bar = 1 μm. (E) Dimensions of a representative cluster (yellow set) and the estimated measurements of a single phage (red set). The values of particle size approximate the expected dimensions of a phage virus labeled by multiple GFP molecules. Scale bar = 0.6 μm. (F) Set of representative macrophages during the uptake of phage particles, SEM scanned at low beam accelerating voltages with SE2 detection at 3 kV acceleration voltage. Scale-bar = 5 μm. (G) A representative macrophage during the uptake of phage particles, in lens SE detection at 3 kV acceleration voltage. Scale bar = 1 μm. (H) Phage particles on silicon, without macrophages, in-lens SE detection (3 kV). Scale bar = 100 nm. (I) Inset showing the uptake of phages into a macrophage, a magnified view of the inset region (red box) in (G) in lens SE detection (3 kV). Scale bar = 100 nm.

Mentions: Phage concentration in the spleen, the major organ responsible for phage clearance2223, revealed key differences between SIR mice and normal control mice (Fig. 1A). In SIR mice, the phage concentration was significantly decreased (2.56-log lower, p < 0.05) in spleen. Intensive clearance of phage was linked to a small but significant decrease (1.14-log, p < 0.05) in the number of phage circulating in the blood of the SIR mice shortly (1 hour) after the phage injection. Other tested organs (lymph nodes, kidneys, muscles, liver) did not reveal significant differences (data not shown), indicating organ-specific clearance activity of SIR macrophages (Fig. 1A). Indeed, phagocytes (splenocytes) from SIR mice tested ex vivo also inactivated the phage more effectively than those isolated from controls (Fig. 1B). Furthermore, we visualized phage degradation by phagocytosis executed by splenocytes, with super-resolution structural-illumination microscope (Fig. 2) and a green fluorescent protein (GFP)-labeled model phage24. The phages were detected within macrophages, typically displayed in groups of GFP-containing particles organized in clusters. The super-resolution imaging was subjected to complementary analysis by spectral unmixing confocal microscopy in lambda model, which was able to identify the pixels of native GFP. This technique showed the ingested phages and co-identified the partially degraded GFP where the pixels displayed red-shifted spectra of GFP (Fig. 2A–E). Thus, we identified phages within macrophage subcellular compartments that were suggestive of the degradation pathway being targeted by bacteriophages in a macrophage.


Mammalian Host-Versus-Phage immune response determines phage fate in vivo.

Hodyra-Stefaniak K, Miernikiewicz P, Drapała J, Drab M, Jończyk-Matysiak E, Lecion D, Kaźmierczak Z, Beta W, Majewska J, Harhala M, Bubak B, Kłopot A, Górski A, Dąbrowska K - Sci Rep (2015)

Visualization of bacteriophage phagocytosis in spleen macrophages up-taking the model phage.The particles of GFP-expressing bacteriophage are visible in discrete locations of the macrophage – as clusters of viral particles. Spectral unmixing confocal microscopy of GFP variants (A,B) powered by super-resolution microscopy (structural illumination mode, (B–D)) of macrophages exposed to phages. A combination of the two complementary imaging modes provides specific information about the presence of native and partially degraded GFP-labeled phages, allows them to be distinguished from the auto-fluorescence background and provides a super-resolved image reconstruction of the phage particle localization pattern. SEM imaging of the phage alone and during phagocytosis by macrophages (F–I). (A) Lambda scan-generated spectra of the enhanced-GFP native form (green curve), the partially degraded form of GFP (red-shifted GFP spectrum, here the red curve), and the auto-fluorescence (magenta curve). (B) Typical morphology of the cell loaded with GFP-labeled phages, spectrally unmixed image showing the native-label particles (green), particles with partially degraded GFP label (red) and auto-fluorescence (magenta). Scale bar = 6 μm (C) Super-resolution imaging of a representative macrophage with the phage particles. Several clusters of particles visible (GFP-labeled phage). Scale bar = 6 μm. (D) Inset showing representative cluster of phages within a macrophage. Scale bar = 1 μm. (E) Dimensions of a representative cluster (yellow set) and the estimated measurements of a single phage (red set). The values of particle size approximate the expected dimensions of a phage virus labeled by multiple GFP molecules. Scale bar = 0.6 μm. (F) Set of representative macrophages during the uptake of phage particles, SEM scanned at low beam accelerating voltages with SE2 detection at 3 kV acceleration voltage. Scale-bar = 5 μm. (G) A representative macrophage during the uptake of phage particles, in lens SE detection at 3 kV acceleration voltage. Scale bar = 1 μm. (H) Phage particles on silicon, without macrophages, in-lens SE detection (3 kV). Scale bar = 100 nm. (I) Inset showing the uptake of phages into a macrophage, a magnified view of the inset region (red box) in (G) in lens SE detection (3 kV). Scale bar = 100 nm.
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Related In: Results  -  Collection

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f2: Visualization of bacteriophage phagocytosis in spleen macrophages up-taking the model phage.The particles of GFP-expressing bacteriophage are visible in discrete locations of the macrophage – as clusters of viral particles. Spectral unmixing confocal microscopy of GFP variants (A,B) powered by super-resolution microscopy (structural illumination mode, (B–D)) of macrophages exposed to phages. A combination of the two complementary imaging modes provides specific information about the presence of native and partially degraded GFP-labeled phages, allows them to be distinguished from the auto-fluorescence background and provides a super-resolved image reconstruction of the phage particle localization pattern. SEM imaging of the phage alone and during phagocytosis by macrophages (F–I). (A) Lambda scan-generated spectra of the enhanced-GFP native form (green curve), the partially degraded form of GFP (red-shifted GFP spectrum, here the red curve), and the auto-fluorescence (magenta curve). (B) Typical morphology of the cell loaded with GFP-labeled phages, spectrally unmixed image showing the native-label particles (green), particles with partially degraded GFP label (red) and auto-fluorescence (magenta). Scale bar = 6 μm (C) Super-resolution imaging of a representative macrophage with the phage particles. Several clusters of particles visible (GFP-labeled phage). Scale bar = 6 μm. (D) Inset showing representative cluster of phages within a macrophage. Scale bar = 1 μm. (E) Dimensions of a representative cluster (yellow set) and the estimated measurements of a single phage (red set). The values of particle size approximate the expected dimensions of a phage virus labeled by multiple GFP molecules. Scale bar = 0.6 μm. (F) Set of representative macrophages during the uptake of phage particles, SEM scanned at low beam accelerating voltages with SE2 detection at 3 kV acceleration voltage. Scale-bar = 5 μm. (G) A representative macrophage during the uptake of phage particles, in lens SE detection at 3 kV acceleration voltage. Scale bar = 1 μm. (H) Phage particles on silicon, without macrophages, in-lens SE detection (3 kV). Scale bar = 100 nm. (I) Inset showing the uptake of phages into a macrophage, a magnified view of the inset region (red box) in (G) in lens SE detection (3 kV). Scale bar = 100 nm.
Mentions: Phage concentration in the spleen, the major organ responsible for phage clearance2223, revealed key differences between SIR mice and normal control mice (Fig. 1A). In SIR mice, the phage concentration was significantly decreased (2.56-log lower, p < 0.05) in spleen. Intensive clearance of phage was linked to a small but significant decrease (1.14-log, p < 0.05) in the number of phage circulating in the blood of the SIR mice shortly (1 hour) after the phage injection. Other tested organs (lymph nodes, kidneys, muscles, liver) did not reveal significant differences (data not shown), indicating organ-specific clearance activity of SIR macrophages (Fig. 1A). Indeed, phagocytes (splenocytes) from SIR mice tested ex vivo also inactivated the phage more effectively than those isolated from controls (Fig. 1B). Furthermore, we visualized phage degradation by phagocytosis executed by splenocytes, with super-resolution structural-illumination microscope (Fig. 2) and a green fluorescent protein (GFP)-labeled model phage24. The phages were detected within macrophages, typically displayed in groups of GFP-containing particles organized in clusters. The super-resolution imaging was subjected to complementary analysis by spectral unmixing confocal microscopy in lambda model, which was able to identify the pixels of native GFP. This technique showed the ingested phages and co-identified the partially degraded GFP where the pixels displayed red-shifted spectra of GFP (Fig. 2A–E). Thus, we identified phages within macrophage subcellular compartments that were suggestive of the degradation pathway being targeted by bacteriophages in a macrophage.

Bottom Line: Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy.We accommodated the experimental data into a mathematical model.We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations.

View Article: PubMed Central - PubMed

Affiliation: Bacteriophage Laboratory, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland.

ABSTRACT
Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy.

No MeSH data available.


Related in: MedlinePlus