The Role of Neutrophils and the Inflammatory Response in Scarring

Heal Ohio 2010
The Role of Neutrophils and the
Inflammatory Response in Scarring
Judith Mack Ph.D and Edward Maytin M.D., Ph.D.

Neutrophil Lineage
Image from: www.autismpedia.org

Mechanisms of neutrophil recruitment to sites of injury
(Leukocyte adhesion cascade)
vesiculo-vacuolar organelles
Adapted from Ley et al., Nature Reviews Immunology 7:678-689 (September 2007)

Dynamics of neutrophil infiltration over the time course of
cutaneus wound healing
lys-EGFP
mouse
Adapted from Kim et al., Journal of Investigate Dermatology 128: 1812-1820 (2008)

Phagocytic and microbicidal activity of neutrophils
Courtesy of DeLeo et al., Infectious Disease Clinics of N.A. 23:17-34, March 2009

Neutrophils are not crucial for normal wound healing?
• 1972 – Simpson and Ross, JCI 51:2009-2023 – depletion of neutrophils
in guinea pigs does not interfere with wound healing (collagen
deposition, wound strength, macrophage infiltration)
• 2003 – Dovi et al., J. Leuk. Biol. 73:448-454 – depletion of neutrophils
in mice does not interfere with wound healing and results in
significant accelerated re-epithelialization

Failure to clear neutrophils is detrimental to healing
Macrophage
Defect or
Depletion
Impaired Wound
Healing and
Increased Scarring
Adapted from Walker et al., Current Drug Targets 4:447-454, 2005

Persistent neutrophil infiltration likely contributes to the
pathophysiology of chronic wounds
Continued exposure to:
• ROS
• Proinflammatory cytokines
• Elastase – increases inflammation, destroys tissue, proteoglycans
and collagen
• Matrix metalloproteases (MMPs) – break down of ECM, growth
factors, and protease inhibitors

Hox Family of Transcription Factors - Hoxb13
Hyaluronan Synathases - Hyaluronan

Hox Transcription Factors Function as
Master Regulators of Differentiation
During Development
Hox transcription factors
Roles of Hox Proteins in Adults
• Angiogenesis
• Cell Proliferation
• Cell Differentiation
• Extracellular Matrix Production (collagen)
• Inflammation
The Atlas of Mouse Development, M. H. Kaufman
Stelnicki et al., JID 111:57-63, 1998 – Hoxb13 is downregulated in fetal skin that
heals without a scar but is not downregulated in adult wounds
Mouse Models
• Hoxb13 KO mouse
• K14-Hoxb13 Transgenic Mouse

Cutaneous wound healing is enhanced in Hoxb13 K0 mice
Mack et al., FASEB J. 17:1351, 2003
blue = collagen

Hoxb13 KO incisional wounds have significantly
greater tensile strength
p

Day
Wound healing is significantly delayed in K14-Hoxb13
transgenic (TG) mice
0 1 3 5 7 9 11
WT
TG3
Mack et al., JID 130:856-65 (March, 2010)

Wound morphology is grossly abnormal in
K14-Hoxb13 TG mice
WTLM
TG6

WTLM
K14-Hoxb13 TG 11-
day-old excisional
wounds display a
protracted neutrophil
presence
TG3
P < 0.05

WTLM
Macrophage counts
are significantly
higher 11-day-old K14-
Hoxb13 TG wounds
TG3
P < 0.005

K14-Hoxb13 TG 11-day-old wounds display a fibrotic
phenotype
WTLM
TG6
Masson’s Trichrome
Blue = collagen

VEGF
TNF-α
TNF-α and VEGF are upregulated in unwounded
and wounded K14-Hoxb13 skin
unwounded
WT TG3 WT TR6
-102-
7-day excisional wound
WT TG3 WT TR6
- 58-
* *
- 43-
- 43-
* *
- 25-
- 37-
GAPDH

Cutaneous wound healing is enhanced in Hoxb13 K0 mice
Mack et al., FASEB J. 17:1351, 2003

Hyaluronan (HA)
HA Synthetic Enzymes
1,4-glucuronic acid
1,3-N-acetylglucosamine
• Hyaluronan Synthase-1 (Has1)
• Hyaluronan Synthase-2 (Has2)
• Hyaluronan Synthase-3 (Has3)
HA Functions
Courtesy of Vincent Hascall., Ph.D. and Torvard Laurent, M. D.
www.glycoforum.gr.jp
• Angiogenesis
• Cell Proliferation
• Cell Differentiation
• Extracellular Matrix Production
• Inflammation
HA Receptors
• CD44
• RHAMM
Mouse Model
• Has1,3 KO mouse

Neutrophil recruitment to the wound site is significantly
increased in Has1/3 KO 1 day-old wounds
WT
Has1,3 KO

HA levels are significantly lower in Has1/3 KO 1-day-old
wounds
*
HA = green
Neuts = red
WT
Has1,3 KO

Final comments
• Neutrophils are critical for eliminating pathogens from wounds.
• Neutrophils are not necessary for the wound repair process.
• The robustness of the neutrophil response may in part be responsible for
the generation of scar tissue in acute and chronic skin wounds.
• Approaches to dampen the neutrophil response may lead to therapies to
reduce scar formation.

Acknowledgements
Cleveland Clinic
Edward V. Maytin, M.D., Ph.D.
Robert Fairchild, Ph.D.
University of Utah, SLC
Mario Capecchi, Ph.D.
Case Western Reserve University
Case Transgenic and Targeting Facility
University of Colorado, Denver
Dr. Xiao-Jing Wang

Neutrophil extracellular nets (NETS) – Another weapon in
the neutrophil aresenal
Nets
• chromatin
• DNA
• granular proteins
Adapted from Brinkmann et al., Science 303:1532-1535 (March 5, 2004)

Fluorophore Assisted Carbohydrate Electrophoresis
•Isolate the HA
•Enzymatically cleave
•Fluorotag disaccharides
•Electrophorese

Cutaneous wound healing is enhanced in Hoxb13 K0 mice
1) Faster epithelial closure
2) Restoration of a more normal collagen architecture
Mack et al., FASEB J. 17:1351, 2003

TNF-α
Mast Cell

Neutrophil Clearance by Macrophages
Image from: www.ruf.rice.edu

Diacovo et al., Journal of Experimental Medicine 202:687-696 (September 5, 2005)

File image: Wikimedia Commons