Neutrophils, not Macrophages, Aid Phage-Mediated Control of Pulmonary Pseudomonas aeruginosa Infection

Chantal Weissfuss^1,2*Karen Hoffmann¹Ulrike Behrendt¹Magdalena Bürkle¹Shailey G. Twamley³Imke H. E. Korf⁴Katharina Ahrens¹Christine Rohde⁵Christian M. Zobel²Laurent Debarbieux⁶Jean-Damien Ricard^7,8Martin Witzenrath^1,9Geraldine Nouailles^1*

• ¹Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Infectious Diseases, Respiratory Medicine and Critical Care, Berlin, Germany
• ²Department of Internal Medicine, Bundeswehrkrankenhaus Berlin, Berlin, Germany
• ³Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Functional Anatomy, Berlin, Germany
• ⁴Pharmaceutical Biotechnology, Fraunhofer Institute for Toxicology and Experimental Medicine, Braunschweig, Germany
• ⁵Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
• ⁶Institut Pasteur, Université Paris Cité, CNRS UMR6047, Department of Microbiology, Bacteriophage Bacteria Host Laboratory, Paris, France
• ⁷Université Paris Cité, Inserm, UMR 1137, Infection Antimicrobials Modelling Evolution, Paris, France
• ⁸Assistance Publique-Hôpitaux de Paris, Hôpital Louis Mourier, DMU ESPRIT, Médecine Intensive Réanimation, Colombes, France
• ⁹German Center for Lung Research (DZL), Berlin, Germany

The increasing prevalence of multidrug-resistant (MDR) bacteria has reduced the effectiveness of standard antibiotics, prompting renewed interest in bacteriophage (phage) therapy as an alternative or adjunctive treatment. Phage therapy offers high specificity, self-amplification at infection sites, and minimal disruption to the gut microbiota. However, clinical implementation is challenging, due to the risk of phage resistance and uncertainties regarding optimal dosing and immune interactions. Previously, we demonstrated that a two-phage cocktail exhibited low immunogenicity in mice and, when combined with meropenem, significantly improved clearance of ventilator-associated Pseudomonas aeruginosa pneumonia, reduced inflammation, and disrupted biofilms more effectively than either treatment alone. In the present study, we investigated the interplay between this phage cocktail and innate immune defenses using a murine respiratory infection model and human in vitro assays. Our findings reveal that the therapeutic efficacy of phage treatment is critically dependent on the presence of neutrophils, which act synergistically with phages to achieve effective bacterial clearance, particularly when bacterial burden exceeds a defined threshold. Alveolar macrophages, however, do not significantly contribute to infection resolution in vivo. Since neutrophils play a key-role in supporting phage-mediated Pseudomonas clearance, the efficacy of phage therapy is closely linked to the hosts immune competence – an important consideration when treating immunocompromised patients.