Journal: Frontiers in Immunology

Article Title: Neutrophils, not macrophages, aid phage-mediated control of pulmonary Pseudomonas aeruginosa infection

doi: 10.3389/fimmu.2025.1681461

Figure Lengend Snippet: Efficacy of opsonophagocytosis is strengthened by phages in vitro . (a) Graphical abstract created with BioRender.com . The in vitro opsonophagocytic killing assay (OPKA) was used for the determination of the phagocytosis rate of differentiated neutrophil-like cells (PMN-dHL-60) or macrophage-like cells (Mϕ-dHL-60). P. aeruginosa (PAO1) were incubated with serial dilutions of anti- Pseudomonas specific antibodies (serum) for 15 min (37 °C, 200 rpm) followed by the addition of baby rabbit complement, dHL-60 cells and the Pseudomonas -specific phage cocktail in indicated concentrations. (b) Transmission electron microscopy visualization shows phage (JG005/JG024; shown by black arrowhead) binding to PAO1 bacteria. Scale bar 50 nm. (c, d) Relative bacterial load (CFU, colony-forming unit) of the OPKA was quantified after 90 min incubation. Samples with dHL-60, serum and complement factors, but no phages serve as (opsono)phagocytosis control. Data determined by 2-way ANOVA with Dunnett´s multiple comparisons test: *p < 0.05; **p < 0.01; n = 4-5. MOI, multiplicity of infection. Pa, P. aeruginosa strain PAO1.

Article Snippet: JG005 (DSM 19872) ( ) , Class: Caudoviricetes Genus: Pakpunavirus Morphology: Myovirus Indicator strain: F2230 Analysis strain: PAO1 (DSM 22644) , 4.30 × 10 1 , DSMZ (Braunschweig, Germany) and Fraunhofer ITEM (Braunschweig, Germany).

Techniques: In Vitro, Incubation, Transmission Assay, Electron Microscopy, Binding Assay, Bacteria, Control, Infection

Journal: Frontiers in Immunology

Article Title: Neutrophils, not macrophages, aid phage-mediated control of pulmonary Pseudomonas aeruginosa infection

doi: 10.3389/fimmu.2025.1681461

Figure Lengend Snippet: In vivo phagocytes depletion strategies. Naive mice (C57BL/6J WT, female, 8–10 weeks; Janvier Labs) were depleted for (a) neutrophils (PMNs) via intraperitoneal (i. p.) application of InVivoPlus anti-mouse Ly6G antibody (clone 1A8; Bio X Cell, Lebanon, USA) or InVivoPlus rat IgG2a, κ isotype control (clone 2A3; Bio X Cell, Lebanon, USA) 24 hours or (e) alveolar macrophages (AlvM) via intratracheal (i. t.) application of Clodronate- or PBS-filled liposomes as control (Liposoma BV, Amsterdam, Netherlands) 72 hours before infection. Experimental plans created with BioRender.com . (b, f) Depletion rates in percent of lungs and BAL cells (BALC). Depletion rates were determined by calculating the ratio of PMNs or AlvM counts in individual depleted animals to the mean PMNs or AlvM count of the corresponding undepleted control group. (c, g) Representative dot blots of the flow cytometry analysis at 24 hours post infection (hpi). Numbers of (d) AlvMs and (h) PMNs determined by flow cytometry. One-way ANOVA, Tukey’s multiple comparisons test. *p < 0,05, **p < 0,01, ***p < 0,001, ****p < 0,0001; depletion control: n = 6 isotype control (d) or n = 6 PBS-liposomes (h), sham-infected, control treated mice (white), depletion controls: n = 6 isotype control (d) or n = 8 PBS-liposomes (h) , PAO1-infected mice with phage treatment (grey, dashed lines), depletion controls: n = 6 isotype control (d) or n = 8 PBS-liposomes (h) , PAO1-infected, control treated mice (grey), n = 5 mice with PMN cell depletion, PAO1-infection and phage treatment (blue, dashed lines), n = 6 mice with PMN depletion, PAO1-infection and control treatment (blue), n = 8 mice with AlvM depletion, PAO1-infection and phage treatment (orange, dashed lines), n = 7 mice with AlvM cell depletion, PAO1-infection and control treatment (orange). BAL, bronchoalveolar lavage.

Article Snippet: JG005 (DSM 19872) ( ) , Class: Caudoviricetes Genus: Pakpunavirus Morphology: Myovirus Indicator strain: F2230 Analysis strain: PAO1 (DSM 22644) , 4.30 × 10 1 , DSMZ (Braunschweig, Germany) and Fraunhofer ITEM (Braunschweig, Germany).

Techniques: In Vivo, Control, Liposomes, Infection, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Neutrophils, not macrophages, aid phage-mediated control of pulmonary Pseudomonas aeruginosa infection

doi: 10.3389/fimmu.2025.1681461

Figure Lengend Snippet: Antimicrobial synergy between phages and innate immune cells. (a) Experimental Plan created with BioRender.com . Naive mice (C57BL/6J WT, female, 8–10 weeks; Janvier Labs) were depleted for alveolar macrophages (AlvM) via intratracheal (i. t.) application of Clodronate- or PBS-filled liposomes as control (Liposoma BV, Amsterdam, Netherlands) 72 hours (h) or neutrophils (PMNs) via intraperitoneal (i. p.) application of InVivoPlus anti-mouse Ly6G antibody (clone 1A8; Bio X Cell, Lebanon, USA) or InVivoPlus rat IgG2a, κ isotype control (clone 2A3; Bio X Cell, Lebanon, USA) 24 h before infection. Mice were intranasally (i. n.) infected with PAO1 (5 × 10 6 /20 µL), while sham-infected mice received PBS as control, followed by one intraperitoneal (i. p.) treatment at 2 h post infection (hpi) with a Pseudomonas -specific phage cocktail (approx. 5 × 10 7 PFU/phage per injection). Analysis time point was 24 hpi. Graphs displaying (b, c) bacterial loads and (d) phage titers at 24 hpi in indicated organs (BAL, homogenized half lungs (right), blood, spleen). Results are shown as box plots depicting median, quartiles, and range. Two-way ANOVA with Tukey’s multiple comparisons test. Significance not shown between PMN and alvM: *p < 0,05, **p < 0,01, ***p < 0,001, ****p < 0,0001; n = 12 control undepleted, sham-infected, control treated mice (white), n = 14 control undepleted, PAO1-infected mice with phage treatment (grey, dashed lines), n = 14 control undepleted, PAO1-infected, control treated mice (grey), n = 5 mice with PMN cell depletion, PAO1-infection and phage treatment (blue, dashed lines), n = 6 mice with PMN depletion, PAO1-infection and control treatment (blue), n = 8 mice with AlvM depletion, PAO1-infection and phage treatment (orange, dashed lines), n = 7 mice with AlvM cell depletion, PAO1-infection and control treatment (orange). Dotted line reflects detection limit. BAL, bronchoalveolar lavage, CFU, colony-forming unit; hpi, hours post infection; PFU, plaque-forming unit.

Article Snippet: JG005 (DSM 19872) ( ) , Class: Caudoviricetes Genus: Pakpunavirus Morphology: Myovirus Indicator strain: F2230 Analysis strain: PAO1 (DSM 22644) , 4.30 × 10 1 , DSMZ (Braunschweig, Germany) and Fraunhofer ITEM (Braunschweig, Germany).

Techniques: Liposomes, Control, Infection, Injection

Journal: Frontiers in Immunology

Article Title: Neutrophils, not macrophages, aid phage-mediated control of pulmonary Pseudomonas aeruginosa infection

doi: 10.3389/fimmu.2025.1681461

Figure Lengend Snippet: Immune cell depletion prior to Pseudomonas infection significantly alters inflammatory cytokine levels (a) Experimental Plan created with BioRender.com . Naive mice (C57BL/6J WT, female, 8–10 weeks; Janvier Labs) were depleted for alveolar macrophages (AlvM) via intratracheal (i. t.) application of Clodronate- or PBS-filled liposomes as control (Liposoma BV, Amsterdam, Netherlands) 72 hours (h) or neutrophils (PMNs) via intraperitoneal (i. p.) application of InVivoPlus anti-mouse Ly6G antibody (clone 1A8; Bio X Cell, Lebanon, USA) or InVivoPlus rat IgG2a, κ isotype control (clone 2A3; Bio X Cell, Lebanon, USA) 24 h before infection. Mice were intranasally (i. n.) infected with PAO1 (5 × 10 6 /20 µL), while sham-infected mice received PBS as control, followed by one intraperitoneal (i. p.) treatment at 2 h post infection (hpi) with a Pseudomonas -specific phage cocktail (approx. 5 × 10 7 PFU/phage per injection). Analysis time point was 24 hpi. Graphs displaying (b) total protein levels (mg/mL) in BAL fluid (BALF) and level of the pro-inflammatory cytokines TNF, IL-6, IL-1β and IL-1α in (c) BALF (ng/mL) or (d) plasma (pg/mL) of Pseudomonas -infected mice. Results are shown as box plots depicting median, quartiles, and range, as determined by ordinary one-way ANOVA with Tukey´s multiple comparisons test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n = 12 control undepleted, sham-infected, control treated mice (white), n = 14 control undepleted, PAO1-infected mice with phage treatment (grey, dashed lines), n = 14 control undepleted, PAO1-infected, control treated mice (grey), n = 5 mice with PMN cell depletion, PAO1-infection and phage treatment (blue, dashed lines), n = 6 mice with PMN depletion, PAO1-infection and control treatment (blue), n = 8 mice with AlvM depletion, PAO1-infection and phage treatment (orange, dashed lines), n = 7 mice with AlvM cell depletion, PAO1-infection and control treatment (orange). BAL, bronchoalveolar lavage; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: JG005 (DSM 19872) ( ) , Class: Caudoviricetes Genus: Pakpunavirus Morphology: Myovirus Indicator strain: F2230 Analysis strain: PAO1 (DSM 22644) , 4.30 × 10 1 , DSMZ (Braunschweig, Germany) and Fraunhofer ITEM (Braunschweig, Germany).

Techniques: Infection, Liposomes, Control, Injection, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Neutrophils, not macrophages, aid phage-mediated control of pulmonary Pseudomonas aeruginosa infection

doi: 10.3389/fimmu.2025.1681461

Figure Lengend Snippet: Neutrophils are essential to control P. aeruginosa infection in mice, regardless of phage treatment. (a) Experimental Plan created with BioRender.com . Naive mice (C57BL/6J WT, female, 8–10 weeks; Janvier Labs) were depleted for alveolar macrophages (AlvM) via intratracheal (i. t.) application of Clodronate- or PBS-filled liposomes as control (Liposoma BV, Amsterdam, Netherlands) 72 hours (h) or neutrophils (PMNs) via intraperitoneal (i. p.) application of InVivoPlus anti-mouse Ly6G antibody (clone 1A8; Bio X Cell, Lebanon, USA) or InVivoPlus rat IgG2a, κ isotype control (clone 2A3; Bio X Cell, Lebanon, USA) 24 h before infection. Mice were intranasally (i. n.) infected with PAO1 (5 × 10 6 /20 µL), while sham-infected mice received PBS as control, followed by one intraperitoneal (i. p.) treatment at 2 h post infection (hpi) with a Pseudomonas -specific phage cocktail (approx. 5 × 10 7 PFU/phage per injection). Analysis time point was 24 hpi. Graphs displaying (b) murine clinical disease score (see ), (c) body weight in percent of pre-infection weight and (d) body temperature (°C) at indicated time points. Results are shown as box plots depicting median, quartiles, and range. Two-way ANOVA with Tukey’s multiple comparisons test. Significance not shown between PMN and alvM: *p < 0,05, **p < 0,01, ***p < 0,001, ****p < 0,0001; n = 12 control undepleted, sham-infected, control treated mice (white), n = 14 control undepleted, PAO1-infected mice with phage treatment (grey, dashed lines), n = 14 control undepleted, PAO1-infected, control treated mice (grey), n = 5 mice with PMN cell depletion, PAO1-infection and phage treatment (blue, dashed lines), n = 6 mice with PMN depletion, PAO1-infection and control treatment (blue), n = 8 mice with AlvM depletion, PAO1-infection and phage treatment (orange, dashed lines), n = 7 mice with AlvM cell depletion, PAO1-infection and control treatment (orange). BAL, bronchoalveolar lavage.

Article Snippet: JG005 (DSM 19872) ( ) , Class: Caudoviricetes Genus: Pakpunavirus Morphology: Myovirus Indicator strain: F2230 Analysis strain: PAO1 (DSM 22644) , 4.30 × 10 1 , DSMZ (Braunschweig, Germany) and Fraunhofer ITEM (Braunschweig, Germany).

Techniques: Control, Infection, Liposomes, Injection