The Effects of Reactive Oxygen Species and Antioxidants on Macrophage Behavior
Robert
0u M H 2O 2
•  Cultured macrophages for 7 days, measured intracellular
ROS at day 7 (n=8)
•  Measure of intracellular production of peroxide
(fluorescent probe is DCFH-DA from Cell Biolabs)
•  M1 and M2c macrophages produce significantly higher
quantities of H2O2 compared to M0 and M2a
macrophages (Fig. 4)
**
**
M0
*
M1
80
RFU
M 2a
60
40
c
M
M
2
a
2
1
M
0
M
0 .0 0 1
AN
AN
IL 1 b
C C L18
C C L22
0 .0 0 0 1
C D 163
CCR7
IL 1 b
10
C C L18
C C L22
C D 163
50u M H 2O 2
***
10
*
**
1
1
0 .1
0 .1
0 .0 1
0 .0 1
•  Culture polarized macrophages on valves, determine which
macrophage phenotype causes highest degree of
degradation
**
0 .0 0 1
0 .0 0 1
IL 1 b
C C L18
C C L22
•
10
0 .1
0 .0 1
0 .0 0 1
IL 1 b
C C L18
C C L22
IL 1 b
C C L18
C C L22
C D 163
IL-1β
(M1
marker)
significantly
decreased at all doses at all time points
Expression of M2a markers increase at
various doses and time points:
o  CCL18 increase after 1uM, 10uM and
50uM treatment
o  CCL22 significantly increases after 24
hours with 1uM treatment
•
** *
1
CCR7
C D 163
C D 163
Fig. 2
1 2 h o u rs p o s t tre a tm e n t
10
10
10
1
1
1
0 .1
0 .1
0 .1
0 .0 1
0 .0 1
0 .0 1
0 .0 0 1
0 .0 0 1
0 .0 0 1
IL 1 b
C C L18
C C L22
10
CCR7
IL 1 b
C C L18
C C L22
C D 163
0uM
100
1uM
10
10uM
50uM
1
1
100uM
0 .1
0 .0 1
0 .1
0 .0 0 0 1
C D 163
6 h r, C C R 7 D o s e R e s p o n s e , D C T
2 4 h o u rs p o s t tre a tm e n t
0 .0 0 1
CCR7
IL 1 b
C C L18
C C L22
C D 163
CCR7
IL 1 b
C C L18
C C L22
C D 163
C
CCR7
Goal
C C R 7 , 1 2 h o u rs p o s t tre a tm e n t
F o ld c h a n g e o v e r G A P D H
1.  Determine the quantity of
ROS produced by the four
different macrophage
phenotypes
2.  Determine the effects of
extracellular ROS on
macrophage polarization
(Fig. 3)
IL 1 b , 6 h o u r s p o s t - t r e a t m e n t
10
IL 1 b , 1 2 h o u r s p o s t t r e a t m e n t
2 .0
**
1 .0
**
1 .5
**
*
0 .8
0 .6
1 .0
0 .4
*
0 .5
0 .2
0 .0
0 .0
C C L 1 8 , 1 2 h o u rs p o s t tre a tm e n t
1
0 .1
100
**
** ****
1
10
0 .0 1
0 .1
0 .0 0 1
1 h r , IL 1 b , D o s e R e s p o n s e , D C T
IL 1 b , 2 4 h o u r s p o s t t r e a t m e n t
0 .8
** ***
**** *
0uM
1uM
0 .6
**
*
References
0 .2
1
0 .0
C D 1 6 3 , 1 2 h o u rs p o s t tre a tm e n t
1
****
0 .1
0 .0 1
****
0 .0 0 0 1
Fig. 6: Data are represented as mean±SEM (One-way ANOVA with Tukey’s post-hoc analysis (n=6)), * p≤ 0.05, ** p ≤.01, *** p ≤.001, ****p ≤ .
0001).
Fig 3.
This project was supported in part by funds from The
Children’s Hospital of Philadelphia, Drexel University, and
Hebrew University of Jerusalem Research Partnership, and
its contents are solely the responsibility of the authors and
do not necessarily represent the official views of The
Children’s Hospital of Philadelphia.
100uM
0 .4
0 .0 0 1
1
10uM
Acknowledgments
50uM
C C L 2 2 , 1 2 h o u rs p o s t tre a tm e n t
**
*
10
F o ld c h a n g e o v e r G A P D H
•
IL-1β
expression
significantly
varied, initially increasing in
expression but after longer treatment
time, decreased in expression
At 12 hours, gene expression of M2a
markers significantly increased while
M1 markers decreased
F o ld c h a n g e o v e r G A P D H
•
F o ld c h a n g e o v e r G A P D H
•  M1 macrophages previously shown to secrete ROS6
•  Production of ROS by other macrophage
phenotypes has not been previously determined
•  Antioxidants have been shown to prevent M0-M2
polarization in mice5
•  Effects have not been determined in human cells
•  Culture macrophages on valves cross-linked with
antioxidant, determine effects on macrophage behavior and
degree of degradation
Fig. 5: Data are represented as mean±SEM (One-way ANOVA with Tukey’s post-hoc analysis (n=6)), * p≤
0.05, ** p ≤.01, *** p ≤.001, ****p ≤ .0001).
7
AN
*
0 .0 0 1
R
AN
M2c
macrophages
0 .0 1
F o ld c h a n g e o v e r G A P D H
IL-10
F o ld c h a n g e o v e r G A P D H
AN
**
0 .0 1
6 h o u rs p o s t tre a tm e n t
1 h o u r p o s t tre a tm e n t
LPS/IFN-g
M2a
macrophages
2 4 h rs
•  Determine effects of antioxidants on macrophage
polarization, add both ROS and antioxidants to determine
changes in gene expression
Dose Response to H2O2
AN
AN
1 2 h rs
**
0 .1
Fig. 4. Data are represented as mean±SEM (**p<0.001, *p<0.05, one-way
ANOVA with Tukey’s post-hoc analysis (n=8).
AN
AN
6 h rs
0 .1
CCR7
AN
IL-4/13
1hr
**
1
100u M H 2O 2
P h e n o ty p e
•  Macrophages are primary cell type recruited to sites of
AN- Apoptotic Neutrophils
inflammation
Neutrophils
•  Macrophages play various roles in N-wound
healing5 (Fig
Injury
2.)
M0
macrophages
** ***
M 2c
0
Monocytes
10
1
Future Experiments
*
**
CCR7
20
Role of Macrophages in
Wound Healing
10
100
10u M H 2O 2
In t r a c e llu la r R O S p r o d u c t io n
100
100
CCR7
side3
•  Valves have been shown to undergo oxidative
degradation, leading to transplant failure after 1-2
years4
•  Source of ROS production unknown
•  Antioxidants added to biomaterial and has proven
effective in preventing degradation4
•  Off-target
effects
of
antioxidants
on
surrounding milieu unknown
M1
macrophages
and Kara L.
1
Spiller
1u M H 2O 2
C
; right: arterial
Gershon
Temporal Response to H2O2
Intracellular Production of
H2O2
F o ld c h a n g e o v e r G A P D H
•  Pediatric heart disease affects ~1 in every 100
newborns1
•  Children’s Hospital of Philadelphia designed
synthetic pulmonary valve to replace dysfunctional
tissue
Fig. 1 Heart valve implants comprised of bovine pericardium (left: ventricular
Kenneth
3
Golomb
University, School of Biomedical Engineering, Science, and Health Systems, 3141 Chesnut Street, Philadelphia, PA 19104
2The Children’s Hospital of Philadelphia, 3401 Civic Center Blvd, Philadelphia, PA, 19104
3The Hebrew University of Jerusalem, 12065 Ein Kerem Medical Center, Jerusalem, Israel, 91120
Introduction
side2
1
Barbee ,
F o ld c h a n g e o v e r G A P D H
1Drexel
2
Levy ,
F o ld c h a n g e o v e r G A P D H
Emily B.
1
Lurier ,
[1] Childrensheartfoundation.org, 'Fact Sheets | The Children’s Heart
Foundation', 2015. [Online]. Available: http://
www.childrensheartfoundation.org/about-chf/fact-sheets. [Accessed: 07- Apr2015].
[2] Labcor.com.br, 2015. [Online]. Available: http://www.labcor.com.br/pub/
301.jpg. [Accessed: 07- Apr- 2015].
[3] Heart-valve-surgery.com, 2015. [Online]. Available: http://www.heart-valvesurgery.com/Images/tissue-valve-replacement-failure-1.jpg. [Accessed: 07- Apr2015].
[4] A. J. Christian, et al., in Biomaterials vol. 35, ed: Elsevier Ltd, 2013, pp.
2097-2102
[5] Y. Zhang, et al., Cell Res vol. 23, ed, 2013, pp. 898-914.
[6] Y. Henrotin, et al., Osteoarthritis and Cartilage vol. 11, ed, 2003, pp.
747-755.