Phages and the immune system – friends or foes?

Ventilator-associated pneumonia (VAP) remains a major clinical challenge, predominantly caused by multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa or Escherichia coli. Since antibiotics are becoming less effective due to the increasing prevalence of MDR infections, bacteriophage (phage) therapy has re-emerged as a promising alternative or adjunct to conventional antimicrobial treatment. However, clinical implementation remains challenging due to critical concerns about phage immunogenicity, limited understanding of phage–host immune interactions, and ongoing doubts about their efficacy and the risk of developing phage resistance. This doctoral thesis focuses on the preclinical evaluation of phage cocktails specifically targeting P. aeruginosa or E. coli, aiming to provide a mechanistic understanding of their therapeutic potential. This work aims at three main aspects: (1) to assess the immunogenicity and systemic safety of the phage cocktails, (2) to evaluate the therapeutic efficacy in a murine VAP model, and (3) to dissect the interplay between phages and innate immune responses. This doctoral research was part of the international MAPVAP consortium (Pre-clinical mechanistic assessment of two bacteriophage cocktails targeting multidrug-resistant Pseudomonas aeruginosa and Escherichia coli for the treatment of ventilator-associated pneumonia), a collaborative project funded by France and Germany of the joint call on antimicrobial resistance. In the first study, we characterized the innate and adaptive immune responses to repeated systemic administration of either the active or UV-inactivated phage cocktails in naïve mice. Despite minor phage-specific humoral responses, neither innate (e.g., monocytes, neutrophils, dendritic cells) nor adaptive (e.g., T cells) immune cells were significantly activated, while the phages reached the lungs. These findings suggest that even repeated systemic administration of phages is associated with only weak immunogenicity and supports the overall safety of phage therapy in immunocompetent hosts in the absence of infection. The second study evaluated the therapeutic efficacy of the P. aeruginosa-specific phage cocktail in comparison to standard antibiotic treatment and a phage-antibiotic combination therapy in a mouse model of Pseudomonas-induced VAP. Combined treatment with phages and meropenem, the standard-of-care antibiotic, significantly enhanced clinical recovery, reduced lung cell damage and improved bacterial clearance. In vitro assays, using primary human air-way epithelial cells, confirmed, that the adjunctive phage therapy lowered the minimum effective concentration of meropenem and delayed the emergence of likely phage-resistant bacterial clones during the treatment. These results highlight the therapeutic potential of combining phages with antibiotics to restore or enhance antimicrobial efficacy in MDR-induced VAP. The third study focused on the interaction between phage therapy and the host’s innate immune response. Using a murine Pseudomonas-respiratory infection model with selected neutrophil or alveolar macrophage depletion, and human in vitro assays, we demonstrated that neutrophils are essential for phage-mediated bacterial clearance. In contrast, alveolar macrophages played a limited role in clearing the infection and immunophage synergy. These results highlight the additive interaction between phages and neutrophils and emphasize the importance of host immune competence for successful phage therapy, especially in immunocompromised individuals. In conclusion, this doctoral thesis provides a detailed analysis of two phage cocktails targeting critical VAP pathogens, confirming their safety regarding immunotolerance, therapeutic potential, especially as an adjunctive treatment and the critical role of the host innate immunity in mediating phage efficacy. Together, these studies support (adjunctive) phage therapy as a promising approach for treating antibiotic-resistant pulmonary infections.