The Role of Neutrophils and the Inflammatory Response in Scarring
The Role of Neutrophils and the Inflammatory Response in Scarring
The Role of Neutrophils and the Inflammatory Response in Scarring
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Heal Ohio 2010<br />
<strong>The</strong> <strong>Role</strong> <strong>of</strong> <strong>Neutrophils</strong> <strong>and</strong> <strong>the</strong><br />
<strong>Inflammatory</strong> <strong>Response</strong> <strong>in</strong> Scarr<strong>in</strong>g<br />
Judith Mack Ph.D <strong>and</strong> Edward Mayt<strong>in</strong> M.D., Ph.D.
Neutrophil L<strong>in</strong>eage<br />
Image from: www.autismpedia.org
Mechanisms <strong>of</strong> neutrophil recruitment to sites <strong>of</strong> <strong>in</strong>jury<br />
(Leukocyte adhesion cascade)<br />
vesiculo-vacuolar organelles<br />
Adapted from Ley et al., Nature Reviews Immunology 7:678-689 (September 2007)
Dynamics <strong>of</strong> neutrophil <strong>in</strong>filtration over <strong>the</strong> time course <strong>of</strong><br />
cutaneus wound heal<strong>in</strong>g<br />
lys-EGFP<br />
mouse<br />
Adapted from Kim et al., Journal <strong>of</strong> Investigate Dermatology 128: 1812-1820 (2008)
Phagocytic <strong>and</strong> microbicidal activity <strong>of</strong> neutrophils<br />
Courtesy <strong>of</strong> DeLeo et al., Infectious Disease Cl<strong>in</strong>ics <strong>of</strong> N.A. 23:17-34, March 2009
<strong>Neutrophils</strong> are not crucial for normal wound heal<strong>in</strong>g?<br />
• 1972 – Simpson <strong>and</strong> Ross, JCI 51:2009-2023 – depletion <strong>of</strong> neutrophils<br />
<strong>in</strong> gu<strong>in</strong>ea pigs does not <strong>in</strong>terfere with wound heal<strong>in</strong>g (collagen<br />
deposition, wound strength, macrophage <strong>in</strong>filtration)<br />
• 2003 – Dovi et al., J. Leuk. Biol. 73:448-454 – depletion <strong>of</strong> neutrophils<br />
<strong>in</strong> mice does not <strong>in</strong>terfere with wound heal<strong>in</strong>g <strong>and</strong> results <strong>in</strong><br />
significant accelerated re-epi<strong>the</strong>lialization
Failure to clear neutrophils is detrimental to heal<strong>in</strong>g<br />
Macrophage<br />
Defect or<br />
Depletion<br />
Impaired Wound<br />
Heal<strong>in</strong>g <strong>and</strong><br />
Increased Scarr<strong>in</strong>g<br />
Adapted from Walker et al., Current Drug Targets 4:447-454, 2005
Persistent neutrophil <strong>in</strong>filtration likely contributes to <strong>the</strong><br />
pathophysiology <strong>of</strong> chronic wounds<br />
Cont<strong>in</strong>ued exposure to:<br />
• ROS<br />
• Pro<strong>in</strong>flammatory cytok<strong>in</strong>es<br />
• Elastase – <strong>in</strong>creases <strong>in</strong>flammation, destroys tissue, proteoglycans<br />
<strong>and</strong> collagen<br />
• Matrix metalloproteases (MMPs) – break down <strong>of</strong> ECM, growth<br />
factors, <strong>and</strong> protease <strong>in</strong>hibitors
Hox Family <strong>of</strong> Transcription Factors - Hoxb13<br />
Hyaluronan Synathases - Hyaluronan
Hox Transcription Factors Function as<br />
Master Regulators <strong>of</strong> Differentiation<br />
Dur<strong>in</strong>g Development<br />
Hox transcription factors<br />
<strong>Role</strong>s <strong>of</strong> Hox Prote<strong>in</strong>s <strong>in</strong> Adults<br />
• Angiogenesis<br />
• Cell Proliferation<br />
• Cell Differentiation<br />
• Extracellular Matrix Production (collagen)<br />
• Inflammation<br />
<strong>The</strong> Atlas <strong>of</strong> Mouse Development, M. H. Kaufman<br />
Stelnicki et al., JID 111:57-63, 1998 – Hoxb13 is downregulated <strong>in</strong> fetal sk<strong>in</strong> that<br />
heals without a scar but is not downregulated <strong>in</strong> adult wounds<br />
Mouse Models<br />
• Hoxb13 KO mouse<br />
• K14-Hoxb13 Transgenic Mouse
Cutaneous wound heal<strong>in</strong>g is enhanced <strong>in</strong> Hoxb13 K0 mice<br />
Mack et al., FASEB J. 17:1351, 2003<br />
blue = collagen
Hoxb13 KO <strong>in</strong>cisional wounds have significantly<br />
greater tensile strength<br />
p
Day<br />
Wound heal<strong>in</strong>g is significantly delayed <strong>in</strong> K14-Hoxb13<br />
transgenic (TG) mice<br />
0 1 3 5 7 9 11<br />
WT<br />
TG3<br />
Mack et al., JID 130:856-65 (March, 2010)
Wound morphology is grossly abnormal <strong>in</strong><br />
K14-Hoxb13 TG mice<br />
WTLM<br />
TG6
WTLM<br />
K14-Hoxb13 TG 11-<br />
day-old excisional<br />
wounds display a<br />
protracted neutrophil<br />
presence<br />
TG3<br />
P < 0.05
WTLM<br />
Macrophage counts<br />
are significantly<br />
higher 11-day-old K14-<br />
Hoxb13 TG wounds<br />
TG3<br />
P < 0.005
K14-Hoxb13 TG 11-day-old wounds display a fibrotic<br />
phenotype<br />
WTLM<br />
TG6<br />
Masson’s Trichrome<br />
Blue = collagen
VEGF<br />
TNF-α<br />
TNF-α <strong>and</strong> VEGF are upregulated <strong>in</strong> unwounded<br />
<strong>and</strong> wounded K14-Hoxb13 sk<strong>in</strong><br />
unwounded<br />
WT TG3 WT TR6<br />
-102-<br />
7-day excisional wound<br />
WT TG3 WT TR6<br />
- 58-<br />
* *<br />
- 43-<br />
- 43-<br />
* *<br />
- 25-<br />
- 37-<br />
GAPDH
Cutaneous wound heal<strong>in</strong>g is enhanced <strong>in</strong> Hoxb13 K0 mice<br />
Mack et al., FASEB J. 17:1351, 2003
Hyaluronan (HA)<br />
HA Syn<strong>the</strong>tic Enzymes<br />
1,4-glucuronic acid<br />
1,3-N-acetylglucosam<strong>in</strong>e<br />
• Hyaluronan Synthase-1 (Has1)<br />
• Hyaluronan Synthase-2 (Has2)<br />
• Hyaluronan Synthase-3 (Has3)<br />
HA Functions<br />
Courtesy <strong>of</strong> V<strong>in</strong>cent Hascall., Ph.D. <strong>and</strong> Torvard Laurent, M. D.<br />
www.glyc<strong>of</strong>orum.gr.jp<br />
• Angiogenesis<br />
• Cell Proliferation<br />
• Cell Differentiation<br />
• Extracellular Matrix Production<br />
• Inflammation<br />
HA Receptors<br />
• CD44<br />
• RHAMM<br />
Mouse Model<br />
• Has1,3 KO mouse
Neutrophil recruitment to <strong>the</strong> wound site is significantly<br />
<strong>in</strong>creased <strong>in</strong> Has1/3 KO 1 day-old wounds<br />
WT<br />
Has1,3 KO
HA levels are significantly lower <strong>in</strong> Has1/3 KO 1-day-old<br />
wounds<br />
*<br />
HA = green<br />
Neuts = red<br />
WT<br />
Has1,3 KO
F<strong>in</strong>al comments<br />
• <strong>Neutrophils</strong> are critical for elim<strong>in</strong>at<strong>in</strong>g pathogens from wounds.<br />
• <strong>Neutrophils</strong> are not necessary for <strong>the</strong> wound repair process.<br />
• <strong>The</strong> robustness <strong>of</strong> <strong>the</strong> neutrophil response may <strong>in</strong> part be responsible for<br />
<strong>the</strong> generation <strong>of</strong> scar tissue <strong>in</strong> acute <strong>and</strong> chronic sk<strong>in</strong> wounds.<br />
• Approaches to dampen <strong>the</strong> neutrophil response may lead to <strong>the</strong>rapies to<br />
reduce scar formation.
Acknowledgements<br />
Clevel<strong>and</strong> Cl<strong>in</strong>ic<br />
Edward V. Mayt<strong>in</strong>, M.D., Ph.D.<br />
Robert Fairchild, Ph.D.<br />
University <strong>of</strong> Utah, SLC<br />
Mario Capecchi, Ph.D.<br />
Case Western Reserve University<br />
Case Transgenic <strong>and</strong> Target<strong>in</strong>g Facility<br />
University <strong>of</strong> Colorado, Denver<br />
Dr. Xiao-J<strong>in</strong>g Wang
Neutrophil extracellular nets (NETS) – Ano<strong>the</strong>r weapon <strong>in</strong><br />
<strong>the</strong> neutrophil aresenal<br />
Nets<br />
• chromat<strong>in</strong><br />
• DNA<br />
• granular prote<strong>in</strong>s<br />
Adapted from Br<strong>in</strong>kmann et al., Science 303:1532-1535 (March 5, 2004)
Fluorophore Assisted Carbohydrate Electrophoresis<br />
•Isolate <strong>the</strong> HA<br />
•Enzymatically cleave<br />
•Fluorotag disaccharides<br />
•Electrophorese
Cutaneous wound heal<strong>in</strong>g is enhanced <strong>in</strong> Hoxb13 K0 mice<br />
1) Faster epi<strong>the</strong>lial closure<br />
2) Restoration <strong>of</strong> a more normal collagen architecture<br />
Mack et al., FASEB J. 17:1351, 2003
TNF-α<br />
Mast Cell
Neutrophil Clearance by Macrophages<br />
Image from: www.ruf.rice.edu
Diacovo et al., Journal <strong>of</strong> Experimental Medic<strong>in</strong>e 202:687-696 (September 5, 2005)
File image: Wikimedia Commons