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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

Innate and adaptive immune response to phage impact phage viability.(A) Tissue concentration of active F8 phage is lowered in mice with a systemic inflammatory response (SIR). SIR was induced in mice (N = 7) by LPS (see: Supplementary). Bacteriophage F8 was injected i.p 109pfu/mouse. Repeated 5 times, exemplary experiment presented, statistics: ANOVA. (B) Activated spleen macrophages neutralize F8 bacteriophage more efficiently. Isolated splenocytes were incubated (N = 5) with F8 phage for 2 h, 37 °C; viable phage was detected in the culture supernatant; ‘splenocytes’- normal splenocytes, ‘SIR splenocytes’- splenocytes from SIR-mice, ‘PMA activated splenocytes’- normal splenocytes induced by phorbolmyristate acetate. Repeated 3 times, exemplary experiment presented, statistics: ANOVA; *p < 0.05, **p < 0.005, ***p < 0.0005. (C) F8 phage induces specific IgM and IgG antibodies in a mouse model. Mice (N = 7) were challenged with F8 subcutaneously 1 × 1010pfu/mouse (control: PBS) on days 0, 20 and 50 (arrows). F8-specific IgM and IgG were detected in sera by ELISA. Repeated 2 times, exemplary experiment presented, statistics: ANOVA. (D) Tissue concentration of active F8 phage is decreased in mice pre-immunized specifically to the phage. F8 phage circulation (3 × 109pfu/mouse i.p.) was compared between pre-immunized and control mice (N = 7). Repeated 3 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005. (E) Specific IgM and IgG antibodies inactivate F8 phage. F8-specific IgM- and IgG-rich serum isolated from mice (N = 7) were incubated for 1.5 hours at 37 °C; phage activity was detected by plating. Repeated 4 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005.
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f1: Innate and adaptive immune response to phage impact phage viability.(A) Tissue concentration of active F8 phage is lowered in mice with a systemic inflammatory response (SIR). SIR was induced in mice (N = 7) by LPS (see: Supplementary). Bacteriophage F8 was injected i.p 109pfu/mouse. Repeated 5 times, exemplary experiment presented, statistics: ANOVA. (B) Activated spleen macrophages neutralize F8 bacteriophage more efficiently. Isolated splenocytes were incubated (N = 5) with F8 phage for 2 h, 37 °C; viable phage was detected in the culture supernatant; ‘splenocytes’- normal splenocytes, ‘SIR splenocytes’- splenocytes from SIR-mice, ‘PMA activated splenocytes’- normal splenocytes induced by phorbolmyristate acetate. Repeated 3 times, exemplary experiment presented, statistics: ANOVA; *p < 0.05, **p < 0.005, ***p < 0.0005. (C) F8 phage induces specific IgM and IgG antibodies in a mouse model. Mice (N = 7) were challenged with F8 subcutaneously 1 × 1010pfu/mouse (control: PBS) on days 0, 20 and 50 (arrows). F8-specific IgM and IgG were detected in sera by ELISA. Repeated 2 times, exemplary experiment presented, statistics: ANOVA. (D) Tissue concentration of active F8 phage is decreased in mice pre-immunized specifically to the phage. F8 phage circulation (3 × 109pfu/mouse i.p.) was compared between pre-immunized and control mice (N = 7). Repeated 3 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005. (E) Specific IgM and IgG antibodies inactivate F8 phage. F8-specific IgM- and IgG-rich serum isolated from mice (N = 7) were incubated for 1.5 hours at 37 °C; phage activity was detected by plating. Repeated 4 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005.

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)

Innate and adaptive immune response to phage impact phage viability.(A) Tissue concentration of active F8 phage is lowered in mice with a systemic inflammatory response (SIR). SIR was induced in mice (N = 7) by LPS (see: Supplementary). Bacteriophage F8 was injected i.p 109pfu/mouse. Repeated 5 times, exemplary experiment presented, statistics: ANOVA. (B) Activated spleen macrophages neutralize F8 bacteriophage more efficiently. Isolated splenocytes were incubated (N = 5) with F8 phage for 2 h, 37 °C; viable phage was detected in the culture supernatant; ‘splenocytes’- normal splenocytes, ‘SIR splenocytes’- splenocytes from SIR-mice, ‘PMA activated splenocytes’- normal splenocytes induced by phorbolmyristate acetate. Repeated 3 times, exemplary experiment presented, statistics: ANOVA; *p < 0.05, **p < 0.005, ***p < 0.0005. (C) F8 phage induces specific IgM and IgG antibodies in a mouse model. Mice (N = 7) were challenged with F8 subcutaneously 1 × 1010pfu/mouse (control: PBS) on days 0, 20 and 50 (arrows). F8-specific IgM and IgG were detected in sera by ELISA. Repeated 2 times, exemplary experiment presented, statistics: ANOVA. (D) Tissue concentration of active F8 phage is decreased in mice pre-immunized specifically to the phage. F8 phage circulation (3 × 109pfu/mouse i.p.) was compared between pre-immunized and control mice (N = 7). Repeated 3 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005. (E) Specific IgM and IgG antibodies inactivate F8 phage. F8-specific IgM- and IgG-rich serum isolated from mice (N = 7) were incubated for 1.5 hours at 37 °C; phage activity was detected by plating. Repeated 4 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594097&req=5

f1: Innate and adaptive immune response to phage impact phage viability.(A) Tissue concentration of active F8 phage is lowered in mice with a systemic inflammatory response (SIR). SIR was induced in mice (N = 7) by LPS (see: Supplementary). Bacteriophage F8 was injected i.p 109pfu/mouse. Repeated 5 times, exemplary experiment presented, statistics: ANOVA. (B) Activated spleen macrophages neutralize F8 bacteriophage more efficiently. Isolated splenocytes were incubated (N = 5) with F8 phage for 2 h, 37 °C; viable phage was detected in the culture supernatant; ‘splenocytes’- normal splenocytes, ‘SIR splenocytes’- splenocytes from SIR-mice, ‘PMA activated splenocytes’- normal splenocytes induced by phorbolmyristate acetate. Repeated 3 times, exemplary experiment presented, statistics: ANOVA; *p < 0.05, **p < 0.005, ***p < 0.0005. (C) F8 phage induces specific IgM and IgG antibodies in a mouse model. Mice (N = 7) were challenged with F8 subcutaneously 1 × 1010pfu/mouse (control: PBS) on days 0, 20 and 50 (arrows). F8-specific IgM and IgG were detected in sera by ELISA. Repeated 2 times, exemplary experiment presented, statistics: ANOVA. (D) Tissue concentration of active F8 phage is decreased in mice pre-immunized specifically to the phage. F8 phage circulation (3 × 109pfu/mouse i.p.) was compared between pre-immunized and control mice (N = 7). Repeated 3 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005. (E) Specific IgM and IgG antibodies inactivate F8 phage. F8-specific IgM- and IgG-rich serum isolated from mice (N = 7) were incubated for 1.5 hours at 37 °C; phage activity was detected by plating. Repeated 4 times, exemplary experiment presented, statistics: ANOVA *p < 0.05, **p < 0.005, ***p < 0.0005.
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