AAC Accepts, published online ahead of print on 4 November 2013 Antimicrob. Agents Chemother. doi:10.1128/AAC.00020-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1
Protective effects of human lactoferrin in Aggregatibacter actinomycetemcomitans
2
induced bacteremia in lactoferrin-deficient mice
3 4
Velusamy, S.K., R. Poojary, R. Ardeshna, Waad Alabdulmohsen, D. H. Fine and K.
5
Velliyagounder*
6
RUTGERS School of Dental Medicine, Newark, New Jersey, USA
7 8 9 10
Running title: Effect of Lactoferrin in A. actinomycetemcomitans bacteremia.
11 12
*Corresponding Author:
13
Kabilan Velliyagounder
14
Department of Oral Biology
15
RUTGERS School of Dental Medicine
16
185 South Orange Ave
17
Newark, New Jersey, 07103, USA
18
Phone: 932-972-5051
19
FAX:
20
Email: [email protected]
973-972-0045
21 22 23
1
24
Abstract
25
Aggregatibacter actinomycetemcomitans, a periodontopathogen has been associated with
26
several systemic diseases. Herein, we report the protective effect of human lactoferrin
27
(hLF) during A. actinomycetemcomitans bacteremia in lactoferrin knockout mice (LFKO-
28
/-
29
cleared the bacteria from blood and organs. Nevertheless, all modes of hLF
30
administration significantly decreased the serum pro-inflammatory cytokines such as
31
IFN- , TNF- , IL-1 , IL-6, IL-10 and IL-12p70 levels. Additionally, hLF administration
32
significantly decreased hepatic and splenic pro-inflammatory cytokine expression levels
33
when compared to the non-hLF treated group. Furthermore, administration of hLF
34
decreased the serum C-reactive protein level, iNOS and MPO gene expression levels in
35
liver and spleen. HLF treatment has also resulted in a six-fold decrease in spleen weight
36
with the migration of typical inflammatory cells in infected mice as a result of decreased
37
inflammatory
38
actinomycetemcomitans bacteremia as indicated by rapid bacterial clearance and
39
decreased host pro-inflammatory mediators.
). The prophylactic, concurrent and therapeutic i.v. administration of hLF significantly
response.
These
results
reveal
that
hLF
protects
against
A.
40 41 42 43 44 45 46
2
47
Introduction
48
Aggregatibacter actinomycetemcomitans is the most extensively reported organism in
49
cases of periodontitis, a chronic inflammatory disease that results in bone resorption,
50
destruction of the connective tissues, and eventual bone loss during later stages of the
51
disease. Oral bacteria very often have the potential to enter into the blood stream as a
52
result of minor trauma including typical daily activities such as brushing teeth and certain
53
invasive medical procedures (1). Therefore, it has been recommended that prophylactic
54
antibiotics be prescribed before medical procedures to limit bacteremia and eventual
55
endocarditis (2).
56 57
The immune system of the host normally protects the body from potentially harmful
58
environmental stimuli by recognizing and responding with multiple immunological
59
reactions. While controlling antigenic stimuli, human lactoferrin (hLF), a major defense
60
protein of the innate immune system also exerts direct first-line defense through its
61
significant impact on the development of adaptive immune responses. The level of the LF
62
is elevated several fold during infection against the antigenic stimuli (3, 4). LF has been
63
isolated primarily from human milk and is secreted by glandular epithelial cells. It is also
64
expressed by immune cells and is notably detected in the secondary granules of
65
neutrophils from which it is released during the inflammatory process. LF also has
66
profound modulatory action on the adaptive immune system by promoting the maturation
67
of T-cell precursors into competent helper cells and the differentiation of immature B-
68
cells into efficient antigen presenting cells (3).
69
3
70
There have been many studies demonstrating the protective effect of LF and LF derived
71
peptides against in vivo infections in wild-type mouse models (Table 1) (5-10).
72
Nonetheless, there is no information about the exclusive protective role of hLF during
73
bacteremia in the absence of endogenous mouse lactoferrin (mLF). We have also
74
demonstrated in our previous study that the LF knockout (LFKO-/-) mice had a higher
75
alveolar bone loss with increased expression of pro-inflammatory cytokines as well as the
76
chemokine expressions during oral infection with A. actinomycetemcomitans. We also
77
reported that the oral infection of A. actinomycetemcomitans resulted in a higher level of
78
systemic dissemination in LFKO-/- mice compared to wild-type mice (11). Further, oral
79
pathogens have the potential to cause frequent bacteremia and it is of our interest to
80
establish a model for LF to control bacteremia in the absence of mLF. Therefore, in the
81
present study, we examined the experimental A. actinomycetemcomitans bacteremia and
82
the eventual patterns of the antimicrobial and immunomodulatory activities with respect
83
to hLF treatment in LFKO-/- mouse model.
84 85
MATERIALS AND METHODS
86
Mice
87
Experimental groups comprised of 7-8 week-old male LFKO-/- mice (12), a generous gift
88
from Dr. Orla Conneely, (Department of Molecular and Cellular Biology, Baylor College
89
of Medicine, Houston, TX, USA) and Dr. David Briles, (Department of Microbiology,
90
University of Alabama at Birmingham, AL, USA). Mice colonies were bred and
91
maintained in the transgenic animal facility of RUTGERS School of Dental Medicine,
92
Newark, New Jersey, USA. The experimental protocol was approved by the campus
4
93
Institutional Animal Care and Use Committee (IACUC) of RUTGERS School of Dental
94
medicine, Newark, NJ, USA.
95 96
Bacterial strain, infection and preparation of hLF
97
A. actinomycetemcomitans CU1000NRif (N=nalidixic acid resistant; Rif=rifampicin
98
resistant), a spontaneous rifampicin resistant strain was used as reported earlier (13). The
99
bacterial count was determined quantitatively, resuspended to 1×107 colony forming unit
100
(CFU) per 0.1 ml of phosphate-buffered saline (PBS) and was i.v. injected into the tail
101
vein. The stock solution of the low endotoxin free, apo-lactoferrin (85-90 % purity)
102
(Sigma Aldrich, St. Louis, MO, USA) prepared in pyrogen-free PBS and filter-sterilized
103
using 0·2 µ acrodisc filters was used for i.v injection or oral feeding without further
104
purification (Gelman Sciences, Ann Arbor, MI, USA). To determine the optimal
105
antimicrobial concentration of hLF against A. actinomycetemcomitans, we used 100-500
106
g/g of mice body weight. For all other further experiments, 300 µg/g mice body weight
107
was used.
108 109
In vivo experimental design
110
The effect of hLF was determined in seven different sets of experiments in LFKO-/- mice
111
which includes; 1) sham infected control mice i.v injected with PBS (control), 2) mice i.v
112
injected with hLF (300 µg/g body weight) (hLF only), 3) hLF (300 µg/g body weight)
113
was
114
actinomycetemcomitans i.v. injected (Oral feeding), 4) mice i.v. injected with A.
115
actinomycetemcomitans (bacteria only), 5) mice i.v injected with hLF and 2 h later, A.
orally
fed
with
a
micro-pipette
one
time
for
three
days
and
A.
5
116
actinomycetemcomitans was i.v. injected (prophylactic), 6) mice i.v injected with A.
117
actinomycetemcomitans and hLF at the same time (concurrent), and 7) mice i.v injected
118
with A. actinomycetemcomitans, and 2 h later hLF was i.v. injected (therapeutic).
119 120
Determination of the viable A. actinomycetemcomitans levels
121
To determine the effective concentration of hLF against bacterial clearance in the blood,
122
different concentration ranging from 100-500 µg/g of body weight were tested. The blood
123
samples were collected at different time intervals by cardiac puncture and serially diluted
124
samples were plated on AAGM plates supplemented with rifampicin (24 µg/ml). Plates
125
were incubated at 37 oC in a 5% CO2 incubator for 48 h and the numbers of colonies
126
enumerated were expressed as CFU/ml of blood. To determine the bacterial count in the
127
organs (brain, heart, kidney, liver, lungs, and spleen) were aseptically removed from the
128
mice and placed in 1 ml of PBS in 50 ml Kendall tissue homogenizers (Tyco Healthcare
129
Group, Mansfield, MA, USA). Following homogenization of the tissues, serially diluted
130
samples were plated on AAGM plates aforesaid. In total, three independent experiments
131
representing three biological replicates were performed, and data were statistically
132
analyzed using one-way ANOVA (13).
133 134
Complete blood count (CBC)
135
Heparinized blood was obtained by retro-orbital phlebotomy under anesthesia and the
136
complete blood count was determined by using an automated H1 Technicon system.
137
(Antech Diagnostics, New Hyde park, NY, USA).
138
6
139
Serum analysis
140
The bioavailability of hLF in mouse serum samples were analyzed using the sandwich
141
enzyme-linked immunosorbent assay (ELISA) as described previously (11). The serum
142
pro-inflammatory
143
Cytokine/chemokine custom multi-cytokine detection and the C-reactive protein levels
144
were analyzed using mouse acute phase kit (Millipore Corporation, Billerica, MA, USA).
145
The Luminex 100 System was used to acquire the results and MILLIPLEX Analyst
146
Software (VigeneTech, Carlisle, MA, USA) was used to analyze the results. Acquisition
147
and analysis have been optimized for multiple parameter measurements as described in
148
the manual.
cytokines
were
analyzed
using
the
MILLIPLEX
mouse
149 150
Real-time RT-PCR gene expression analysis
151
The extraction of total RNA was carried out using an RNeasy Mini Kit (Qiagen Inc,
152
Valencia, CA, USA). Real time PCR analysis was carried out according to the
153
manufacturer’s protocol (High capacity cDNA reverse transcription kit; Fast SYBR
154
Green Master Mix, ABI, Foster City, CA, USA). The primer sequences and conditions
155
for the hepatic and splenic cytokine expression levels were followed as reported earlier
156
(11). Primers used for the expression analysis of the inducible nitric oxide synthase
157
(iNOS) and myeloperoxidase (MPO) are given in Table 2. Results are the mean ± SEM
158
of the duplicate experiments of three independent samples.
159 160
Histopathology of spleen
161
At 48 h post-infection, aseptic collection of spleen was performed as described above.
7
162
The tissues were fixed in 4 % Para formaldehyde for 48 h. Tissue sections were routinely
163
processed, embedded in paraffin, mounted on slides, and stained with hematoxylin and
164
eosin (H&E) (14). Tissue sections were observed under Olympus light microscope at
165
×1,000 magnification.
166 167
Statistical analysis
168
Significance in the differences between the groups was calculated using Student’s t test.
169
Values of P
1
Protective effects of human lactoferrin in Aggregatibacter actinomycetemcomitans
2
induced bacteremia in lactoferrin-deficient mice
3 4
Velusamy, S.K., R. Poojary, R. Ardeshna, Waad Alabdulmohsen, D. H. Fine and K.
5
Velliyagounder*
6
RUTGERS School of Dental Medicine, Newark, New Jersey, USA
7 8 9 10
Running title: Effect of Lactoferrin in A. actinomycetemcomitans bacteremia.
11 12
*Corresponding Author:
13
Kabilan Velliyagounder
14
Department of Oral Biology
15
RUTGERS School of Dental Medicine
16
185 South Orange Ave
17
Newark, New Jersey, 07103, USA
18
Phone: 932-972-5051
19
FAX:
20
Email: [email protected]
973-972-0045
21 22 23
1
24
Abstract
25
Aggregatibacter actinomycetemcomitans, a periodontopathogen has been associated with
26
several systemic diseases. Herein, we report the protective effect of human lactoferrin
27
(hLF) during A. actinomycetemcomitans bacteremia in lactoferrin knockout mice (LFKO-
28
/-
29
cleared the bacteria from blood and organs. Nevertheless, all modes of hLF
30
administration significantly decreased the serum pro-inflammatory cytokines such as
31
IFN- , TNF- , IL-1 , IL-6, IL-10 and IL-12p70 levels. Additionally, hLF administration
32
significantly decreased hepatic and splenic pro-inflammatory cytokine expression levels
33
when compared to the non-hLF treated group. Furthermore, administration of hLF
34
decreased the serum C-reactive protein level, iNOS and MPO gene expression levels in
35
liver and spleen. HLF treatment has also resulted in a six-fold decrease in spleen weight
36
with the migration of typical inflammatory cells in infected mice as a result of decreased
37
inflammatory
38
actinomycetemcomitans bacteremia as indicated by rapid bacterial clearance and
39
decreased host pro-inflammatory mediators.
). The prophylactic, concurrent and therapeutic i.v. administration of hLF significantly
response.
These
results
reveal
that
hLF
protects
against
A.
40 41 42 43 44 45 46
2
47
Introduction
48
Aggregatibacter actinomycetemcomitans is the most extensively reported organism in
49
cases of periodontitis, a chronic inflammatory disease that results in bone resorption,
50
destruction of the connective tissues, and eventual bone loss during later stages of the
51
disease. Oral bacteria very often have the potential to enter into the blood stream as a
52
result of minor trauma including typical daily activities such as brushing teeth and certain
53
invasive medical procedures (1). Therefore, it has been recommended that prophylactic
54
antibiotics be prescribed before medical procedures to limit bacteremia and eventual
55
endocarditis (2).
56 57
The immune system of the host normally protects the body from potentially harmful
58
environmental stimuli by recognizing and responding with multiple immunological
59
reactions. While controlling antigenic stimuli, human lactoferrin (hLF), a major defense
60
protein of the innate immune system also exerts direct first-line defense through its
61
significant impact on the development of adaptive immune responses. The level of the LF
62
is elevated several fold during infection against the antigenic stimuli (3, 4). LF has been
63
isolated primarily from human milk and is secreted by glandular epithelial cells. It is also
64
expressed by immune cells and is notably detected in the secondary granules of
65
neutrophils from which it is released during the inflammatory process. LF also has
66
profound modulatory action on the adaptive immune system by promoting the maturation
67
of T-cell precursors into competent helper cells and the differentiation of immature B-
68
cells into efficient antigen presenting cells (3).
69
3
70
There have been many studies demonstrating the protective effect of LF and LF derived
71
peptides against in vivo infections in wild-type mouse models (Table 1) (5-10).
72
Nonetheless, there is no information about the exclusive protective role of hLF during
73
bacteremia in the absence of endogenous mouse lactoferrin (mLF). We have also
74
demonstrated in our previous study that the LF knockout (LFKO-/-) mice had a higher
75
alveolar bone loss with increased expression of pro-inflammatory cytokines as well as the
76
chemokine expressions during oral infection with A. actinomycetemcomitans. We also
77
reported that the oral infection of A. actinomycetemcomitans resulted in a higher level of
78
systemic dissemination in LFKO-/- mice compared to wild-type mice (11). Further, oral
79
pathogens have the potential to cause frequent bacteremia and it is of our interest to
80
establish a model for LF to control bacteremia in the absence of mLF. Therefore, in the
81
present study, we examined the experimental A. actinomycetemcomitans bacteremia and
82
the eventual patterns of the antimicrobial and immunomodulatory activities with respect
83
to hLF treatment in LFKO-/- mouse model.
84 85
MATERIALS AND METHODS
86
Mice
87
Experimental groups comprised of 7-8 week-old male LFKO-/- mice (12), a generous gift
88
from Dr. Orla Conneely, (Department of Molecular and Cellular Biology, Baylor College
89
of Medicine, Houston, TX, USA) and Dr. David Briles, (Department of Microbiology,
90
University of Alabama at Birmingham, AL, USA). Mice colonies were bred and
91
maintained in the transgenic animal facility of RUTGERS School of Dental Medicine,
92
Newark, New Jersey, USA. The experimental protocol was approved by the campus
4
93
Institutional Animal Care and Use Committee (IACUC) of RUTGERS School of Dental
94
medicine, Newark, NJ, USA.
95 96
Bacterial strain, infection and preparation of hLF
97
A. actinomycetemcomitans CU1000NRif (N=nalidixic acid resistant; Rif=rifampicin
98
resistant), a spontaneous rifampicin resistant strain was used as reported earlier (13). The
99
bacterial count was determined quantitatively, resuspended to 1×107 colony forming unit
100
(CFU) per 0.1 ml of phosphate-buffered saline (PBS) and was i.v. injected into the tail
101
vein. The stock solution of the low endotoxin free, apo-lactoferrin (85-90 % purity)
102
(Sigma Aldrich, St. Louis, MO, USA) prepared in pyrogen-free PBS and filter-sterilized
103
using 0·2 µ acrodisc filters was used for i.v injection or oral feeding without further
104
purification (Gelman Sciences, Ann Arbor, MI, USA). To determine the optimal
105
antimicrobial concentration of hLF against A. actinomycetemcomitans, we used 100-500
106
g/g of mice body weight. For all other further experiments, 300 µg/g mice body weight
107
was used.
108 109
In vivo experimental design
110
The effect of hLF was determined in seven different sets of experiments in LFKO-/- mice
111
which includes; 1) sham infected control mice i.v injected with PBS (control), 2) mice i.v
112
injected with hLF (300 µg/g body weight) (hLF only), 3) hLF (300 µg/g body weight)
113
was
114
actinomycetemcomitans i.v. injected (Oral feeding), 4) mice i.v. injected with A.
115
actinomycetemcomitans (bacteria only), 5) mice i.v injected with hLF and 2 h later, A.
orally
fed
with
a
micro-pipette
one
time
for
three
days
and
A.
5
116
actinomycetemcomitans was i.v. injected (prophylactic), 6) mice i.v injected with A.
117
actinomycetemcomitans and hLF at the same time (concurrent), and 7) mice i.v injected
118
with A. actinomycetemcomitans, and 2 h later hLF was i.v. injected (therapeutic).
119 120
Determination of the viable A. actinomycetemcomitans levels
121
To determine the effective concentration of hLF against bacterial clearance in the blood,
122
different concentration ranging from 100-500 µg/g of body weight were tested. The blood
123
samples were collected at different time intervals by cardiac puncture and serially diluted
124
samples were plated on AAGM plates supplemented with rifampicin (24 µg/ml). Plates
125
were incubated at 37 oC in a 5% CO2 incubator for 48 h and the numbers of colonies
126
enumerated were expressed as CFU/ml of blood. To determine the bacterial count in the
127
organs (brain, heart, kidney, liver, lungs, and spleen) were aseptically removed from the
128
mice and placed in 1 ml of PBS in 50 ml Kendall tissue homogenizers (Tyco Healthcare
129
Group, Mansfield, MA, USA). Following homogenization of the tissues, serially diluted
130
samples were plated on AAGM plates aforesaid. In total, three independent experiments
131
representing three biological replicates were performed, and data were statistically
132
analyzed using one-way ANOVA (13).
133 134
Complete blood count (CBC)
135
Heparinized blood was obtained by retro-orbital phlebotomy under anesthesia and the
136
complete blood count was determined by using an automated H1 Technicon system.
137
(Antech Diagnostics, New Hyde park, NY, USA).
138
6
139
Serum analysis
140
The bioavailability of hLF in mouse serum samples were analyzed using the sandwich
141
enzyme-linked immunosorbent assay (ELISA) as described previously (11). The serum
142
pro-inflammatory
143
Cytokine/chemokine custom multi-cytokine detection and the C-reactive protein levels
144
were analyzed using mouse acute phase kit (Millipore Corporation, Billerica, MA, USA).
145
The Luminex 100 System was used to acquire the results and MILLIPLEX Analyst
146
Software (VigeneTech, Carlisle, MA, USA) was used to analyze the results. Acquisition
147
and analysis have been optimized for multiple parameter measurements as described in
148
the manual.
cytokines
were
analyzed
using
the
MILLIPLEX
mouse
149 150
Real-time RT-PCR gene expression analysis
151
The extraction of total RNA was carried out using an RNeasy Mini Kit (Qiagen Inc,
152
Valencia, CA, USA). Real time PCR analysis was carried out according to the
153
manufacturer’s protocol (High capacity cDNA reverse transcription kit; Fast SYBR
154
Green Master Mix, ABI, Foster City, CA, USA). The primer sequences and conditions
155
for the hepatic and splenic cytokine expression levels were followed as reported earlier
156
(11). Primers used for the expression analysis of the inducible nitric oxide synthase
157
(iNOS) and myeloperoxidase (MPO) are given in Table 2. Results are the mean ± SEM
158
of the duplicate experiments of three independent samples.
159 160
Histopathology of spleen
161
At 48 h post-infection, aseptic collection of spleen was performed as described above.
7
162
The tissues were fixed in 4 % Para formaldehyde for 48 h. Tissue sections were routinely
163
processed, embedded in paraffin, mounted on slides, and stained with hematoxylin and
164
eosin (H&E) (14). Tissue sections were observed under Olympus light microscope at
165
×1,000 magnification.
166 167
Statistical analysis
168
Significance in the differences between the groups was calculated using Student’s t test.
169
Values of P
