Background: Premature neonates who develop pneumonia (PNA) have worse pulmonary & neurodevelopmental outcomes. Previous studies suggest multiple functional defects in the neonate that increase susceptibility to PNA: including reduced neutrophil mobilization, complement activation, & bacterial clearing. Our goal is to deliver molecules, such as anti-microbials, to neutrophils to overcome some of these functional defects. We chose a biocompatible nanoparticle, dextran-lysozyme nanogels (NG), which has been shown in our lab to be taken up by neutrophils in a murine in vivo inflammation model, in vitro human neutrophils, & in ex-vivo human lungs. We hypothesize serum opsonization enhances phagocytic entry of NG to murine neutrophils.
Objective: To determine if serum opsonization enhances uptake of NG by murine neutrophils, & by what mechanism.
Design/Methods: Neutrophils were isolated from wild type murine bone marrow by negative selection. FITC-NG were exposed to mouse serum for 1h at 37C. Purified neutrophils were exposed to either naive NG or serum-incubated NG at 37C. Complement action was blocked by: heat treatment, which denatures complement proteins, & cobra venom factor, which depletes C3. For heat treatment, mouse serum was heated to 56C x 1 hour. Cobra venom factor (CVF) was incubated with mouse serum at 37C x 1 hour. FITC-NG uptake was quantified by flow cytometry. The same serum-incubation protocol was followed for FITC-NG, as well as control particle: adenovirus, & then particles were washed & pelleted for proteomic analysis by mass spectrometry.
Results: In vitro NG-neutrophil interaction increased following serum exposure quantified by both the percent positive neutrophils & the mean fluorescence intensity (MFI) of neutrophils. Serum exposure more than doubled the percent of neutrophils containing NG & caused more than a 10-fold increase in the MFI. The increased interaction was abrogated by both heat-treatment & CVF-treatment of serum. Serum-opsonized NGs had a preponderance of C3 peptides bound to them, which were not seen in naïve NG or similarly serum exposed adenovirus particles.
Conclusions: This study demonstrates that neutrophils phagocytose NG particles & serum exposure increases both the number of neutrophils taking up particles, as well as the number of particles per neutrophil. The serum effect was blocked by complement denaturation & C3 depletion, demonstrating the necessity of complement to efficient phagocytosis of particles. The presence of C3 opsonization was confirmed by proteomic analysis. We speculate that NG loaded with anti-microbial molecules will co-localize with bacteria within phagocytes, potentially improving bacterial clearance by neutrophils. Due to their functionally deficient neutrophils & relative complement deficiency, bioengineered particles with C3 present on the surface which will improve bacteria killing & clearance via anti-microbial loaded-NG will be particularly beneficial in the neonatal population.
