Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils

Abstract

Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the innate host response, and their recruitment to the lungs and subsequent ubiquitous phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary disease is characterized by progressive pulmonary decline governed by a persistent, exaggerated inflammatory response dominated by PMNs. The principal contributor is chronic Pseudomonas aeruginosa biofilm infection, which attracts and activates PMNs and thereby is responsible for the continuing inflammation. Strategies to prevent initial airway colonization with P. aeruginosa by augmenting the phagocytic competence of PMNs may postpone the deteriorating chronic biofilm infection. Anti-P. aeruginosa IgY antibodies significantly increase the PMN-mediated respiratory burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is attributed to IgY-facilitated formation of immobilized bacteria in aggregates, as visualized by fluorescence microscopy and the induction of increased bacterial hydrophobicity. Thus, the present study demonstrates that avian egg yolk immunoglobulins (IgY) targeting P. aeruginosa modify bacterial fitness, which enhances bacterial killing by PMN-mediated phagocytosis and thereby may facilitate a rapid bacterial clearance in airways of people with cystic fibrosis.

FIG 1

Respiratory burst assay results. The production of reactive oxygen was detected by luminol-enhanced chemiluminescence during phagocytosis of PAO1 cells by PMNs. The chemiluminescence, given in counts per second (CPS), was recorded using luminol and a microplate fluorescence reader (Wallac 1420 Victor2; PerkinElmer) during 60 min of phagocytosis. Each panel shows the cumulative CPS ± the standard error of the mean of experiments run in duplicate. *, lowest concentration of S-IgY to give a significant increase in respiratory burst, compared to the non-IgY control (P < 0.003).

FIG 2

The consequence of Fc receptor blocking on the cumulative burst of PMNs phagocytizing S-IgY-, C-IgY-, or IgG-opsonized PAO1 cells. Fc receptors of PMNs were blocked with FcγRI MAb (anti-CD64), FcγRII MAb (anti-CD16), and FcγRIII MAb (anti-CD32) prior to phagocytosis. The chemiluminescence, given in counts per second (CPS), was determined by using luminol in a microplate fluorescence reader (Wallac 1420 Victor2; PerkinElmer) during 60 min of phagocytosis. Each panel shows the cumulative CPS ± the standard error of the mean of experiments run in duplicate from three different donors. *, blocking Fc receptors (CD16, CD32, and CD64) significantly reduced the cumulative chemiluminescence detected when PMNs phagocytized IgG-opsonized bacteria (P < 0.001). **, no significant difference in chemiluminescence was detected when PMNs phagocytized S-IgY-opsonized PAO1 cells.

FIG 3

PMN-mediated bacterial killing, defined as the proportion of viable bacteria after 30 min of phagocytosis compared to S-IgY-opsonized controls. Isolated PMNs were challenged with S-IgY- or C-IgY-opsonized or non-IgY (-IgY) PAO1 cells, and the number of viable bacteria was determined by colony counting the next day. Each bar shows the percent survival ± the standard error of the mean of experiments run in duplicates. *, P < 0.05; **, P < 0.03.

FIG 4

Different images from indirect immunofluorescence microscopy of PAO1 opsonized with S-IgY. PAO1 was opsonized with 10% S-IgY and allowed to aggregate for 60 min at 37°C prior to addition of Texas Red-conjugated rabbit anti-chicken IgG secondary antibody. The top images show the Texas Red-fluorescent anti-chicken IgG-detecting S-IgY antibodies. The bottom images display the corresponding DAPI-stained images.

FIG 5

The time-dependent development of S-IgY-mediated aggregates of PAO1 (GFP). (Upper left) Dispersed bacteria prior to addition of S-IgY. (Upper right) Formation of bacterial aggregates 30 min after S-IgY was added. (Bottom) Close-ups of bacterial aggregates 2 h after S-IgY addition. The bottom right image shows the non-IgY control after 2 h.

FIG 6

The distribution of mean particle size, measured by fluorescence-activated cell sorting. PAO1 was opsonized with S-IgY or C-IgY and allowed to aggregate for 60 min prior to determining the particle size distribution (based on FSC-A). *, P < 0.01; **, P < 0.05.

FIG 7

Still images from time-lapse microscopy of PMNs phagocytizing PAO1 cells during a time course of 20 min. S-IgY-opsonized PAO1 cells (GFP) were allowed to aggregate for 1 h at 37°C prior to addition of PMNs. Arrows display bacterial aggregates becoming internalized by PMNs.

FIG 8

The MATH test was applied to evaluate bacterial hydrophobicity. After thoroughly mixing PAO1 and the hydrocarbon n-hexane, a two-phase separation was established and the difference in the OD pre- and postmixing is expressed as the hydrophobicity index. Thus, a reduction of the OD in the aqueous phase after mixing, e.g., bacteria were localized in the n-hexane phase, increased the HI. PAO1 prior to mixing with n-hexane was opsonized by S-IgY, C-IgY, or IgG. Results are depicted as the HI ± the standard error of the mean from experiments run in duplicate. *, P < 0.004; **, P < 0.002; ***, P < 0.02.