IAI Accepted Manuscript Posted Online 20 April 2015 Infect. Immun. doi:10.1128/IAI.02970-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 2 3 4 5 6 7 8 9 10 11 12
Anti-Pseudomonas aeruginosa IgY antibodies induce specific bacterial aggregation and internalization in human polymorphonuclear neutrophils K. Thomsen1, L. Christophersen1, T. Bjarnsholt1,2, P.Ø. Jensen1, C. Moser1, N. Høiby1,2. 1
Department of Clinical Microbiology, Rigshospitalet, Copenhagen University Hospital, Denmark.
2
Department of International Health, Immunology and Microbiology, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark
13 14 15
Correspondence: Niels Høiby, Department of Clinical Microbiology, Rigshospitalet, Copenhagen University Hospital, Juliane Maries Vej 22, Copenhagen, Denmark. Email: [email protected]
16
Keywords: Cystic fibrosis, Pseudomonas aeruginosa, IgY, Phagocytosis
17 18 19 20
Abstract
21 22
Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the
23
innate host response and their recruitment to the lungs and subsequent ubiquitous
24
phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary
25
disease is characterized by progressive pulmonary decline governed by a persistent,
26
exaggerated inflammatory response dominated by PMNs. The principal contributor is
27
chronic Pseudomonas aeruginosa biofilm infection, which attracts and activates
28
PMNs and thereby is responsible for the continuing inflammation. Strategies to
29
prevent initial airway colonization with P. aeruginosa by augmenting the phagocytic
30
competence of PMNs may postpone the deterioating chronic biofilm infection. Anti-
31
P. aeruginosa IgY antibodies significantly increase the PMN mediated respiratory
32
burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is
33
attributed to IgY facilitated formation of immobilized bacteria in aggregates as
34
visualized by fluorescent microscopy and the induction of increased bacterial
35
hydrophobicity. Thus Present study demonstrates that avian egg yolk
36
immunoglobulins (IgY) targeting P. aeruginosa modifies bacterial fitness, which
37
enhances bacterial killing by PMN mediated phagocytosis and thereby may facilitate
38
a rapid bacterial clearance in the cystic fibrosis airways.
39 40 41
Introduction
42 43
Innate immunity is vital for controlling primary infection in the respiratory tract.
44
Activation of cellular constituents in the innate host response promotes development
45
and differentiation of adaptive host mechanisms and the subsequent synergistic
46
interplay eliminates encountered pathogens and establishes long-lasting protective
47
immunity (1). Polymorphonuclear neutrophils (PMNs) are essential determinants in
48
the innate host response and are readily recruited to the site of infection. Their
49
immune response is activated in part by bacterial shedding of immuno-stimulatory
50
pathogen-associated molecular patterns (PAMPs), like lipopolysaccharide (LPS),
51
DNA, cell-wall components and flagella that are recognized by epithelial pattern
52
recognition receptors (PRRs) such as Toll-like receptors (TLRs), C-type lectin
53
receptors and the cytoplasmic NOD-like receptors (NLRs) (2,3,4). The stimulation of
54
PRRs activates downstream pathway signalling through an adaptor molecule,
55
MyD88, leading to nuclear translocation of the transcription factor nuclear factor κΒ
56
(NF-κΒ) (5). NF-κΒ activates gene promoters controlling a broad range of cytokines
57
and initiates the expression of proinflammatory effectors. The subsequent expression
58
of tumor necrosis factor-α (TNF-α) up-regulates the cell-adhesion molecule ICAM-1
59
on epithelial cells, which is the ligand for β2 integrin on PMNs, priming the
60
extravasation of PMNs (6) to the alveolar lumen, where the cells eventually
61
commence their bactericidal task of phagocytizing and killing pathogens.
62
Phagocytosis is a sequential process involving recognition of damaging pathogens,
63
continued by attachment, engulfment and degradation. The phagocytic process is
64
greatly enhanced by bacterial opsonization, especially with IgG and fragments of
65
complement effector C3 (7). The engagement of phagocyte receptors and opsonized
66
bacteria activates cytoskeletal contractile components, causing invagination of the
67
membrane and extension of pseudopods around the microbe. The consecutive
68
interplay of receptor-opsonin pairs conducts the engulfment of bacteria within a
69
phagosome, leading to formation of the phagolysosome by fusion of the phagosome
70
and lysosomal compartments containing bactericidal products. The bactericidal
71
mechanisms of PMNs are thus characterized by the production of antimicrobial
72
metabolites such as peptides, proteases and reactive oxygen species (ROS) during
73
phagocytosis (8). Phagocytosis terminates by degradation of microbes and the
74
apoptotic consequence of PMNs and subsequent engulfment by macrophages,
75
initiates the resolution of inflammation (9).
76
Cystic fibrosis (CF) pulmonary disease is characterized by prominent airway
77
inflammation as evidenced by PMN accumulation and excessive concentrations of
78
the neutrophil chemokine IL-8 (10,11,12). The sustained PMN activation produces
79
tissue destructive components like neutrophil elastase (13), proteases (14) and
80
reactive oxygen species (ROS), which contribute to the pulmonary disease by tissue
81
degradation (15). The deterioating chronic airway inflammation is attributed to
82
recurring bacterial colonization, which eventually progress into chronic infection due
83
to failure of eradicating bacteria e.g. due to biofilm formation. The normal cessation
84
of inflammation is annulled and the PMNs are arrested in an accelerated state
85
aggravating the destruction of lung tissue further reinforcing inflammatory responses.
86
P. aeruginosa is the predominant bacterial pathogen in CF and the opportunistic
87
pathogen readily adapts to the mucus-rich environment in the CF lung (16). Chronic
88
infection with P. aeruginosa is associated with decline in lung function and frequent
89
exacerbations (17) and early colonization with P. aeruginosa is a predictor of poor
90
prognosis (18). The initial colonization of planktonic P. aeruginosa is eradicated
91
efficiently by competent PMNs (19). However, recurrent colonization triggers
92
bacterial adaptation to the airway milieu causing a shift from planktonic state to
93
biofilm mode of growth and the selection for bacterial mutants with abundant
94
production of the exopolysaccharide alginate (20), thereby establishing mucoid
95
phenotypes which are resilient to phagocytosis (21, 22). Thus approaches to moderate
96
the innate host response at early stage of CF disease, prior to development of chronic
97
infection, by improving the phagocytic character of PMNs may assist current
98
antibiotic treatment regimens in reducing P. aeruginosa colonization in non-
99
chronically infected patients. Passive immunotherapy is a potent and promising
100
adjuvant to standard therapy against infectious diseases (23). Egg yolk
101
immunoglobulins (IgY) have been used successfully to eradicate infectious diseases
102
in animals (24) and prophylaxis with egg yolk immunoglobulins (IgY) targeting P.
103
aeruginosa reduces colonization in CF patients (25). The encouraging results from
104
clinical studies on the efficacy of IgY immunotherapy to prevent gastrointestinal
105
infections (26) and animal models showing favourable impact of IgY therapy on
106
influenza virus (27,28), contribute to the potential benefit of anti-P. aeruginosa IgY
107
prophylaxis in non-chronically infected CF patients. Thus entering pathogens in the
108
respiratory airways of CF patients may be readily killed by PMNs due to moderation
109
of the phagocytic activity mediated by IgY immunotherapy. In the present study we
110
describe that IgY opsonisation induces increased hydrophobicity and aggregation of
111
P. aeruginosa, which facilitates augmented phagocytic activity of PMNs and
112
subsequent bacterial killing.
113 114 115 116 117
Materials and Methods
118 119
Anti-Pseudomonas aeruginosa IgY: Solution containing specific anti-P. aeruginosa
120
(S-IgY) or non-specific (C-IgY) IgY antibodies were obtained from ImmunSystem
121
I.M.S. (Uppsala, Sweden). The anti-P. aeruginosa IgY antibodies employed in these
122
experiments are harvested from egg yolk of White leghorn chickens immunized with
123
six different P. aeruginosa O-antigen strains (O1, O3, O5, O6, O9, O11).
124 125
P. aeruginosa PAO1 vaccine strain was grown in LB broth. From a frozen stock an
126
overnight culture was prepared and re-cultured in LB to obtain an OD600=0.5. The log
127
phase bacteria were washed twice in PBS before application.
128 129
Neutrophil isolation: Human PMNs were harvested from healthy donors by density
130
gradient centrifugation. Erythrocytes were allowed to sediment in 5% dextran and the
131
leucocyte-enriched plasma was layered on Lymphoprep (Axis-Shield, Norway) and
132
centrifuged at 2200g for 15 min. at room temperature. The supernatant were
133
discarded and the remaining erythrocytes within the pelleted PMNs were removed by
134
hypertonic lysis. The purified PMNs were resuspended in Krebs-Ringer buffer
135
supplemented with 10 mM glucose (KRB).
136 137
Respiratory burst assay: The production of reactive oxygen species (ROS) during
138
oxidative burst was measured by luminol-enhanced chemiluminescense. PAO1 was
139
opsonized by S-IgY or C-IgY for 60 min at 37°C on a platform rocker to allow
140
aggregation prior to application. For control experiments PAO1 was opsonized by
141
human AB positive serum and treated similarly to IgY opsonized organisms. Isolated
142
PMNs at a density of 5 x 106 cells/ml were mixed with pre-opsonized bacteria (5 x
143
107 cfu/ml) in a 96-well microtiter plate (Nunc). Luminol was finally added to the
144
wells and the luminol-enhanced chemiluminescence detecting phagocyte-derived
145
reactive oxygen species (ROS) was recorded every second minute using a
146
luminometer (Wallac 1420 Victor2, Perkin Elmer) at 37°C for 1 hour.
147 148
Bactericidal assay: The killing of pre-opsonized bacteria by PMNs was performed
149
in polypropylene tubes with a final volume of 600 μl/tube according to the protocol
150
described by Green and colleagues (29). Briefly, isolated PMNs (5 x 106 cells/ml)
151
were mixed with PAO1 (5 x 107 cfu/ml) resulting in a bacteria-to-PMN ratio of 10 to
152
1. The bacteria were prior to application opsonized with 10% S-IgY, C-IgY or human
153
AB positive serum for 60 min at 37°C on a platform rocker. At time-points 0 and 30
154
min post-mixing PMNs and PAO1, 50 µl of the reaction mixture was removed and
155
added to 2.45 ml H2O pH 11, initiating cellular lysis for 5 min before vigorously
156
vortexing and plating onto modified Conradi-Drigalski plates (State Seruminstitute,
157
Copenhagen, Denmark), incubated overnight at 37°C. Colony counting the next day
158
determined the PMN mediated bacterial killing by the loss of bacterial viability
159
during 30 min of phagocytosis compared to S-IgY opsonized controls.
160 161
Neutrophil receptor blockage: To examine the role of Fc-receptor mediated
162
phagocytosis, human Fc receptors (FcγRI, FcγRII, FcγRIII) were blocked by FcγRI
163
mAbs (anti-CD64), FcγRII mAbs (anti-CD16) and FcγRIII mAbs (anti-CD32)
164
antibodies (R&D Systems, Minneapolis, USA). Freshly isolated PMNs (5 x 106/ml)
165
were pretreated with 0.5 µg of anti-CD16, anti-CD32 or anti-CD64 for 15 min at
166
37°C followed by the detection of ROS production by luminol chemiluminescense
167
assay as previously described where PMNs were allowed to phagocytize PAO1 pre-
168
opsonized with S-IgY, C-IgY or IgG.
169 170
Bacterial hydrophobicity: The partitioning of aqueous and hydrocarbon phases in
171
the microbial adhesion to hydrocarbons (MATH) test was utilized to determine
172
hydrophobicity. The affinity of bacteria to hexane in a water-hexane two-phase
173
system as described by Rosenberg et al was applied (30). Washed PAO1 were pre-
174
opsonized with S-IgY, C-IgY or IgG and suspended in 3 ml PBS to an OD600 of 1.0.
175
A volume of 0.5 ml n-hexane was carefully layered on top of the bacterial suspension
176
in disposable glass tubes. The tube contents were vortexed for 60 s and after phase
177
separation occurred, the OD of the aqueous phase was measured. The relative
178
bacterial hydrophobicity was expressed as a hydrophobicity index (HI):
179
HI = (Initial absorbance – absorbance after phase separation) / Initial absorbance.
180
Accordingly hydrophobic bacteria will have lower scores than hydrophilic organisms.
181 182
Flowcytometry: Fluorescent PAO1 (tagged with green fluorescent protein [GFP])
183
were mixed with either S-IgY or C-IgY and allowed to aggregate at room
184
temperature for 60 min before estimation of size according to the forward light-
185
scatter (FSC-A) with a FACScanto (BD biosciences). Bacteria were discriminated
186
according to green fluorescence intensity collected in FL-1 and at least 10,000 events
187
were recorded for each sample. Cytometer Setup and Tracking Beads (BD
188
biosciences) were used for instrument calibration and flow data was processed and
189
analyzed by Diva (BD biosciences)
190 191
Indirect immunofluorescence microscopy: PAO1 vaccine strain cultured in LB
192
medium was washed twice in PBS and mixed with 10% S-IgY at 37°C for 1 h on a
193
platform rocker. After washing twice with PBS, TexasRed-conjugated rabbit anti-
194
chicken IgG secondary antibody (Abcam plc, Cambridge, UK), diluted 1:500 with
195
PBS containing 1% bovine albumin serum (BAS), was added and incubated at 37°C
196
for 1 h on a platform rocker. After washing twice with PBS, aliquots were smeared
197
on microscope slide and air-dried followed by a drop of DAPI stain and mounting of
198
coverslip. Immunofluorescent micrographs of specimens were obtained using a
199
fluorescence microscope.
200 201
Bacterial internalization by PMN: The uptake of bacteria in PMNs was examined
202
by confocal scanning laser microscopy (CSLM) (Zeiss Imager.Z2, LSM 710 CLSM)
203
and the accompanying software (Zeiss Zen 2010 v. 6.0.; Zeiss, Germany). Overnight
204
cultures of fluorescent PAO1 (green fluorescent protein [GFP]) were adjusted to a
205
density of 108 CFU/ml in PBS. Aliquots of PAO1 (50 μl) were opsonized with 10%
206
S-IgY in a 96-well microtiter plate and kept at room temperature for 2 hours to allow
207
aggregation. Phagocytosis was initiated by addition of freshly isolated PMNs (107
208
cells/ml) in KRB to the bacterial suspension and the bacterial internalization by
209
PMNs was visualized after incubation for 30 min at 37°C and 200 rpm.
210 211 212 213 214 215
Results
216 217 218
The respiratory burst produced by PMNs phagocytizing P. aeruginosa (PAO1)
219
is significantly increased by S-IgY.
220
Reactive oxygen species (ROS) is produced by PMNs phagocytosis of bacteria
221
during the respiratory burst. Luminol-enhanced chemiluminescense assay quantify
222
the ROS production by luminescence when activated by oxidants produced during
223
the respiratory burst. Figure 1 shows the chemiluminescense detected during the
224
phagocytosis of P. aeruginosa PAO1 by PMNs. The bacteria are preopsonized with
225
serum and increasing concentrations of S-IgY, C-IgY or PBS (non-IgY) as control. S-
226
IgY augments the chemiluminescense in a concentration-dependent manner and the
227
lowest concentration of S-IgY tested (0.1%) significantly increases the
228
chemiluminescense compared to non-IgY (p
1 2 3 4 5 6 7 8 9 10 11 12
Anti-Pseudomonas aeruginosa IgY antibodies induce specific bacterial aggregation and internalization in human polymorphonuclear neutrophils K. Thomsen1, L. Christophersen1, T. Bjarnsholt1,2, P.Ø. Jensen1, C. Moser1, N. Høiby1,2. 1
Department of Clinical Microbiology, Rigshospitalet, Copenhagen University Hospital, Denmark.
2
Department of International Health, Immunology and Microbiology, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark
13 14 15
Correspondence: Niels Høiby, Department of Clinical Microbiology, Rigshospitalet, Copenhagen University Hospital, Juliane Maries Vej 22, Copenhagen, Denmark. Email: [email protected]
16
Keywords: Cystic fibrosis, Pseudomonas aeruginosa, IgY, Phagocytosis
17 18 19 20
Abstract
21 22
Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the
23
innate host response and their recruitment to the lungs and subsequent ubiquitous
24
phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary
25
disease is characterized by progressive pulmonary decline governed by a persistent,
26
exaggerated inflammatory response dominated by PMNs. The principal contributor is
27
chronic Pseudomonas aeruginosa biofilm infection, which attracts and activates
28
PMNs and thereby is responsible for the continuing inflammation. Strategies to
29
prevent initial airway colonization with P. aeruginosa by augmenting the phagocytic
30
competence of PMNs may postpone the deterioating chronic biofilm infection. Anti-
31
P. aeruginosa IgY antibodies significantly increase the PMN mediated respiratory
32
burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is
33
attributed to IgY facilitated formation of immobilized bacteria in aggregates as
34
visualized by fluorescent microscopy and the induction of increased bacterial
35
hydrophobicity. Thus Present study demonstrates that avian egg yolk
36
immunoglobulins (IgY) targeting P. aeruginosa modifies bacterial fitness, which
37
enhances bacterial killing by PMN mediated phagocytosis and thereby may facilitate
38
a rapid bacterial clearance in the cystic fibrosis airways.
39 40 41
Introduction
42 43
Innate immunity is vital for controlling primary infection in the respiratory tract.
44
Activation of cellular constituents in the innate host response promotes development
45
and differentiation of adaptive host mechanisms and the subsequent synergistic
46
interplay eliminates encountered pathogens and establishes long-lasting protective
47
immunity (1). Polymorphonuclear neutrophils (PMNs) are essential determinants in
48
the innate host response and are readily recruited to the site of infection. Their
49
immune response is activated in part by bacterial shedding of immuno-stimulatory
50
pathogen-associated molecular patterns (PAMPs), like lipopolysaccharide (LPS),
51
DNA, cell-wall components and flagella that are recognized by epithelial pattern
52
recognition receptors (PRRs) such as Toll-like receptors (TLRs), C-type lectin
53
receptors and the cytoplasmic NOD-like receptors (NLRs) (2,3,4). The stimulation of
54
PRRs activates downstream pathway signalling through an adaptor molecule,
55
MyD88, leading to nuclear translocation of the transcription factor nuclear factor κΒ
56
(NF-κΒ) (5). NF-κΒ activates gene promoters controlling a broad range of cytokines
57
and initiates the expression of proinflammatory effectors. The subsequent expression
58
of tumor necrosis factor-α (TNF-α) up-regulates the cell-adhesion molecule ICAM-1
59
on epithelial cells, which is the ligand for β2 integrin on PMNs, priming the
60
extravasation of PMNs (6) to the alveolar lumen, where the cells eventually
61
commence their bactericidal task of phagocytizing and killing pathogens.
62
Phagocytosis is a sequential process involving recognition of damaging pathogens,
63
continued by attachment, engulfment and degradation. The phagocytic process is
64
greatly enhanced by bacterial opsonization, especially with IgG and fragments of
65
complement effector C3 (7). The engagement of phagocyte receptors and opsonized
66
bacteria activates cytoskeletal contractile components, causing invagination of the
67
membrane and extension of pseudopods around the microbe. The consecutive
68
interplay of receptor-opsonin pairs conducts the engulfment of bacteria within a
69
phagosome, leading to formation of the phagolysosome by fusion of the phagosome
70
and lysosomal compartments containing bactericidal products. The bactericidal
71
mechanisms of PMNs are thus characterized by the production of antimicrobial
72
metabolites such as peptides, proteases and reactive oxygen species (ROS) during
73
phagocytosis (8). Phagocytosis terminates by degradation of microbes and the
74
apoptotic consequence of PMNs and subsequent engulfment by macrophages,
75
initiates the resolution of inflammation (9).
76
Cystic fibrosis (CF) pulmonary disease is characterized by prominent airway
77
inflammation as evidenced by PMN accumulation and excessive concentrations of
78
the neutrophil chemokine IL-8 (10,11,12). The sustained PMN activation produces
79
tissue destructive components like neutrophil elastase (13), proteases (14) and
80
reactive oxygen species (ROS), which contribute to the pulmonary disease by tissue
81
degradation (15). The deterioating chronic airway inflammation is attributed to
82
recurring bacterial colonization, which eventually progress into chronic infection due
83
to failure of eradicating bacteria e.g. due to biofilm formation. The normal cessation
84
of inflammation is annulled and the PMNs are arrested in an accelerated state
85
aggravating the destruction of lung tissue further reinforcing inflammatory responses.
86
P. aeruginosa is the predominant bacterial pathogen in CF and the opportunistic
87
pathogen readily adapts to the mucus-rich environment in the CF lung (16). Chronic
88
infection with P. aeruginosa is associated with decline in lung function and frequent
89
exacerbations (17) and early colonization with P. aeruginosa is a predictor of poor
90
prognosis (18). The initial colonization of planktonic P. aeruginosa is eradicated
91
efficiently by competent PMNs (19). However, recurrent colonization triggers
92
bacterial adaptation to the airway milieu causing a shift from planktonic state to
93
biofilm mode of growth and the selection for bacterial mutants with abundant
94
production of the exopolysaccharide alginate (20), thereby establishing mucoid
95
phenotypes which are resilient to phagocytosis (21, 22). Thus approaches to moderate
96
the innate host response at early stage of CF disease, prior to development of chronic
97
infection, by improving the phagocytic character of PMNs may assist current
98
antibiotic treatment regimens in reducing P. aeruginosa colonization in non-
99
chronically infected patients. Passive immunotherapy is a potent and promising
100
adjuvant to standard therapy against infectious diseases (23). Egg yolk
101
immunoglobulins (IgY) have been used successfully to eradicate infectious diseases
102
in animals (24) and prophylaxis with egg yolk immunoglobulins (IgY) targeting P.
103
aeruginosa reduces colonization in CF patients (25). The encouraging results from
104
clinical studies on the efficacy of IgY immunotherapy to prevent gastrointestinal
105
infections (26) and animal models showing favourable impact of IgY therapy on
106
influenza virus (27,28), contribute to the potential benefit of anti-P. aeruginosa IgY
107
prophylaxis in non-chronically infected CF patients. Thus entering pathogens in the
108
respiratory airways of CF patients may be readily killed by PMNs due to moderation
109
of the phagocytic activity mediated by IgY immunotherapy. In the present study we
110
describe that IgY opsonisation induces increased hydrophobicity and aggregation of
111
P. aeruginosa, which facilitates augmented phagocytic activity of PMNs and
112
subsequent bacterial killing.
113 114 115 116 117
Materials and Methods
118 119
Anti-Pseudomonas aeruginosa IgY: Solution containing specific anti-P. aeruginosa
120
(S-IgY) or non-specific (C-IgY) IgY antibodies were obtained from ImmunSystem
121
I.M.S. (Uppsala, Sweden). The anti-P. aeruginosa IgY antibodies employed in these
122
experiments are harvested from egg yolk of White leghorn chickens immunized with
123
six different P. aeruginosa O-antigen strains (O1, O3, O5, O6, O9, O11).
124 125
P. aeruginosa PAO1 vaccine strain was grown in LB broth. From a frozen stock an
126
overnight culture was prepared and re-cultured in LB to obtain an OD600=0.5. The log
127
phase bacteria were washed twice in PBS before application.
128 129
Neutrophil isolation: Human PMNs were harvested from healthy donors by density
130
gradient centrifugation. Erythrocytes were allowed to sediment in 5% dextran and the
131
leucocyte-enriched plasma was layered on Lymphoprep (Axis-Shield, Norway) and
132
centrifuged at 2200g for 15 min. at room temperature. The supernatant were
133
discarded and the remaining erythrocytes within the pelleted PMNs were removed by
134
hypertonic lysis. The purified PMNs were resuspended in Krebs-Ringer buffer
135
supplemented with 10 mM glucose (KRB).
136 137
Respiratory burst assay: The production of reactive oxygen species (ROS) during
138
oxidative burst was measured by luminol-enhanced chemiluminescense. PAO1 was
139
opsonized by S-IgY or C-IgY for 60 min at 37°C on a platform rocker to allow
140
aggregation prior to application. For control experiments PAO1 was opsonized by
141
human AB positive serum and treated similarly to IgY opsonized organisms. Isolated
142
PMNs at a density of 5 x 106 cells/ml were mixed with pre-opsonized bacteria (5 x
143
107 cfu/ml) in a 96-well microtiter plate (Nunc). Luminol was finally added to the
144
wells and the luminol-enhanced chemiluminescence detecting phagocyte-derived
145
reactive oxygen species (ROS) was recorded every second minute using a
146
luminometer (Wallac 1420 Victor2, Perkin Elmer) at 37°C for 1 hour.
147 148
Bactericidal assay: The killing of pre-opsonized bacteria by PMNs was performed
149
in polypropylene tubes with a final volume of 600 μl/tube according to the protocol
150
described by Green and colleagues (29). Briefly, isolated PMNs (5 x 106 cells/ml)
151
were mixed with PAO1 (5 x 107 cfu/ml) resulting in a bacteria-to-PMN ratio of 10 to
152
1. The bacteria were prior to application opsonized with 10% S-IgY, C-IgY or human
153
AB positive serum for 60 min at 37°C on a platform rocker. At time-points 0 and 30
154
min post-mixing PMNs and PAO1, 50 µl of the reaction mixture was removed and
155
added to 2.45 ml H2O pH 11, initiating cellular lysis for 5 min before vigorously
156
vortexing and plating onto modified Conradi-Drigalski plates (State Seruminstitute,
157
Copenhagen, Denmark), incubated overnight at 37°C. Colony counting the next day
158
determined the PMN mediated bacterial killing by the loss of bacterial viability
159
during 30 min of phagocytosis compared to S-IgY opsonized controls.
160 161
Neutrophil receptor blockage: To examine the role of Fc-receptor mediated
162
phagocytosis, human Fc receptors (FcγRI, FcγRII, FcγRIII) were blocked by FcγRI
163
mAbs (anti-CD64), FcγRII mAbs (anti-CD16) and FcγRIII mAbs (anti-CD32)
164
antibodies (R&D Systems, Minneapolis, USA). Freshly isolated PMNs (5 x 106/ml)
165
were pretreated with 0.5 µg of anti-CD16, anti-CD32 or anti-CD64 for 15 min at
166
37°C followed by the detection of ROS production by luminol chemiluminescense
167
assay as previously described where PMNs were allowed to phagocytize PAO1 pre-
168
opsonized with S-IgY, C-IgY or IgG.
169 170
Bacterial hydrophobicity: The partitioning of aqueous and hydrocarbon phases in
171
the microbial adhesion to hydrocarbons (MATH) test was utilized to determine
172
hydrophobicity. The affinity of bacteria to hexane in a water-hexane two-phase
173
system as described by Rosenberg et al was applied (30). Washed PAO1 were pre-
174
opsonized with S-IgY, C-IgY or IgG and suspended in 3 ml PBS to an OD600 of 1.0.
175
A volume of 0.5 ml n-hexane was carefully layered on top of the bacterial suspension
176
in disposable glass tubes. The tube contents were vortexed for 60 s and after phase
177
separation occurred, the OD of the aqueous phase was measured. The relative
178
bacterial hydrophobicity was expressed as a hydrophobicity index (HI):
179
HI = (Initial absorbance – absorbance after phase separation) / Initial absorbance.
180
Accordingly hydrophobic bacteria will have lower scores than hydrophilic organisms.
181 182
Flowcytometry: Fluorescent PAO1 (tagged with green fluorescent protein [GFP])
183
were mixed with either S-IgY or C-IgY and allowed to aggregate at room
184
temperature for 60 min before estimation of size according to the forward light-
185
scatter (FSC-A) with a FACScanto (BD biosciences). Bacteria were discriminated
186
according to green fluorescence intensity collected in FL-1 and at least 10,000 events
187
were recorded for each sample. Cytometer Setup and Tracking Beads (BD
188
biosciences) were used for instrument calibration and flow data was processed and
189
analyzed by Diva (BD biosciences)
190 191
Indirect immunofluorescence microscopy: PAO1 vaccine strain cultured in LB
192
medium was washed twice in PBS and mixed with 10% S-IgY at 37°C for 1 h on a
193
platform rocker. After washing twice with PBS, TexasRed-conjugated rabbit anti-
194
chicken IgG secondary antibody (Abcam plc, Cambridge, UK), diluted 1:500 with
195
PBS containing 1% bovine albumin serum (BAS), was added and incubated at 37°C
196
for 1 h on a platform rocker. After washing twice with PBS, aliquots were smeared
197
on microscope slide and air-dried followed by a drop of DAPI stain and mounting of
198
coverslip. Immunofluorescent micrographs of specimens were obtained using a
199
fluorescence microscope.
200 201
Bacterial internalization by PMN: The uptake of bacteria in PMNs was examined
202
by confocal scanning laser microscopy (CSLM) (Zeiss Imager.Z2, LSM 710 CLSM)
203
and the accompanying software (Zeiss Zen 2010 v. 6.0.; Zeiss, Germany). Overnight
204
cultures of fluorescent PAO1 (green fluorescent protein [GFP]) were adjusted to a
205
density of 108 CFU/ml in PBS. Aliquots of PAO1 (50 μl) were opsonized with 10%
206
S-IgY in a 96-well microtiter plate and kept at room temperature for 2 hours to allow
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aggregation. Phagocytosis was initiated by addition of freshly isolated PMNs (107
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cells/ml) in KRB to the bacterial suspension and the bacterial internalization by
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PMNs was visualized after incubation for 30 min at 37°C and 200 rpm.
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Results
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The respiratory burst produced by PMNs phagocytizing P. aeruginosa (PAO1)
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is significantly increased by S-IgY.
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Reactive oxygen species (ROS) is produced by PMNs phagocytosis of bacteria
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during the respiratory burst. Luminol-enhanced chemiluminescense assay quantify
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the ROS production by luminescence when activated by oxidants produced during
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the respiratory burst. Figure 1 shows the chemiluminescense detected during the
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phagocytosis of P. aeruginosa PAO1 by PMNs. The bacteria are preopsonized with
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serum and increasing concentrations of S-IgY, C-IgY or PBS (non-IgY) as control. S-
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IgY augments the chemiluminescense in a concentration-dependent manner and the
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lowest concentration of S-IgY tested (0.1%) significantly increases the
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chemiluminescense compared to non-IgY (p
