Researcher(s)
- Adhya Anilkumar, Biomedical Engineering, University of Delaware
Faculty Mentor(s)
- Catherine Fromen, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Lower respiratory infections (LRIs) remain a leading global health burden, with bacterial infiltration and biofilms that can limit antibiotic efficacy. Bacteriophages are a promising alternative to antibiotics, capable of lysing bacterial pathogens. Their nanoscale size enables deposition in the lower respiratory tract and interaction with innate immune cells. However, the development of inhalable phage therapies is underexplored due to limited understanding of phage-lung interactions.
To evaluate T4 phage interactions with macrophages, crude T4 phage lysates were purified using spin columns and 1-octanol organic phase extraction. Endotoxin concentrations were quantified via Limulus Amebocyte Lysate assay, and phage DNA concentrations were measured by absorbance spectroscopy. SYBR Gold-stained phages were visualized in RAW 264.7 macrophages via fluorescence microscopy. Macrophage metabolic activity post-phage treatment was measured using Cell Titer-Glo. Flow cytometry was conducted to assess macrophage polarization and immune response.
Both purification methods reduced the endotoxin concentration by 10,000-fold using spin columns and 26-fold using organic extraction. However, the spin column purification resulted in a 3-fold reduction in phage DNA, suggesting a loss of phages. RAW 264.7 cells treated with purified lysates demonstrated significantly higher metabolic activity at 24 hours compared to crude lysate and LPS controls, indicating that purified phages are non-cytotoxic to macrophage function. The crude and purified T4 phages were then co-administered with lipopolysaccharide (LPS) to mimic an LRI inflammatory environment. Macrophages dosed with purified T4 phages expressed minimal CD40+, a pro-inflammatory marker, compared to crude T4 phage lysate and LPS. Maximal phage internalization and CD40+ expression were observed at 24 hours, followed by phage clearance by 72 hours, corresponding with a resolution of inflammation. These findings demonstrate that endotoxin-free phage samples are able to exhibit dynamic immunomodulatory effects in macrophages. Future work will examine the pathways by which macrophages internalize phages and how phages interact with various macrophage cell lines.