Transcript S. aureus infection

Alteration in cytokine
and chemokine
expression during
Staphylococcus aureus
wound infections
By: Kayla Bounds
Definition of a wound
• A wound is the
disruption of normal
anatomic structure and
function
Lazarus, G.S., et al., 1994. 130(4): p. 489-93
Stages of wound healing
Time line for wound healing on a log scale
7-300 days
1-30 days (peak 7 days)
0.15-10 day (peak 1 day)
0-0.1 day
Cytokines
• What is a cytokine?
• Proteins that are important for cell- to- cell signaling and
the initiation of the immune response
• Cytokines and wound healing
• In normal wound healing, cytokines will recruit
keratinocytes, fibroblasts, endothelial cells,
macrophages, and platelets
• In chronic wounds, pro-inflammatory cytokines recruit
neutrophils
• This results in a state of constant inflammation, preventing
proper wound healing
Acute wounds
• Proceed normally through an orderly and timely
regenerative process that results in restoration of
anatomic function and integrity
http://emedicine.medscape.com/artic
le/1277941-overview#aw2aab6b5
Lazarus, G.S., et al., Definitions and guidelines for assessment of wounds and evaluation of
healing. Arch Dermatol, 1994. 130(4): p. 489-93
Chronic wounds
• Chronic wounds fail to proceed through an orderly
and timely process to produce anatomic and
functional integrity
• Usually remain in the inflammation phase of the
healing process
Diabetic foot ulcer
Biofilmbook.hypertextbookshop.com
Venous leg ulcer
Biofilmbook.hypertextbookshop.com
Pressure ulcer
Biofilmbook.hypertextbookshop.com
Wound colonization
1° colonizers:
Gram positive cocci
S. aureus, S. epidermidis,
Streptococcus spp., Enterococcus spp.
2 ° colonizers:
Gram negative rods
P. aeruginosa, E. coli,
K. pneumoniae, A. baumannii
Wound bed
3 ° colonizers:
Anaerobic species
Prevotella spp., Porphorymonas spp.
Biofilm within wounds
Dirckx, P. Center for BiofilmEngineering. Montana State University
Hypothesis
• Bacterial infection of wounds significantly alter the
expression of cytokines and chemokines
Objectives
1. To generate a wound using the murine model of
wound infection
2. Infect the wound with S. aureus
3. Examine the expression of cytokines 1 day, 3 day,
and 7 day post infection
Staphylococcus aureus
• Catalase-positive,
gram- positive
• Cocci arranged in
clusters
• Facultative
anaerobe
• Capsule and slime
layer
• Commonly
isolated in the
nasal passage
Berger, J. 2011. Science Photo Lib.
Murray, P. et al. 2005.
Murine Model of Wound Infection
Remove
hair from
mice
Euthanize
mice and
collect
tissue
1.5 x 1.5 cm full-thickness
surgical wound covered with
OPSITE dressing
Inject of S. aureus under
dressing
Experimental design 1
Uninjured
Injured uninfected
Collect the tissues 1 day post
injury/infected
Injured infected
Experimental design 2
5 mm-tissue punch
from site adjacent to
wound
RNA later
Homogenize
tissue in RNA
Bee
Extract RNA
Reverse
transcribe
to cDNA
Use data for
analysis
Perform real-time PCR
using RT² Profiler™ PCR
Array Mouse Cytokines
and Chemokines kit
(QIAGEN)
Perform initial realtime PCR using
primers for specific
cytokines to check
RNA quality
Cytokines/chemokines and the
functional groups
Response to injury
Immune
Responses
Hematopoiesis
and Production
of immune cells
Recruitment,
activation
of immune cells
Response to injury
Injured/uninjured
fold change
1.58
Protein
Function
Adipoq
Metabolism (fat cells)
Bmp2
Bone mineralization
1.50
Bmp4
Bone formation
2.00
Bmp6
Response to injury
Osteoclast Function, wound
healing
Central Nervous system
Wound repair, matrix maintenance,
fibrosis
Wound healing
1.59
Spp1
Cntf
Tgfb
Ccl20
31.96
3.14
1.00
63.70
Response to injury
Injured infected/Injured
uninfected fold change
-3.23
Protein
Function
Adipoq
Metabolism (fat cells)
Bmp2
Bone mineralizaiton
-4.00
Bmp4
Bone formation
-3.23
Bmp6
Response to injury
-4.00
Spp1
Osteoclast function, wound healing
-4.00
Cntf
Central nervous system
Wound repair, matrix maintenance,
fibrosis
Wound healing
-4.00
Tgfb
Ccl-20
-4.00
-1.27
Recruitment and activation of
immune cells
Injured/Uninjured
fold change
18.45
Protein
Function
Csf3
G-Csf: activation of granulocytes
Cxcl1
PMN chemotaxis
43.91
Cxcl3
PMN chemotaxis
193.55
Cxcl5
PMN chemotaxis
2198.70
Ppbp
CXCL-7: PMN chemotaxis
20.43
Ccl2
MCP-1: Monocyte chemotaxis, differentiation
12.60
Ccl3
MIP-1a:Monocyte chemotaxis, macrophage diff.
133.56
Ccl4
MIP-1b: Monocyte chemotaxis, macrophage diff.
100.17
Ccl7
MCP-3: Monocyte chemotaxis, macrophage diff.
6.32
Ltb
TNFb :Lymphocyte development in periphery
-2.00
Cxcl10
Th1, CD8, NK cell movement
-1.28
Ccl17
Th2, Treg trafficking
1.57
Cxcl13
B cell movement
10.02
Recruitment and activation of
immune cells
Protein
Function
Csf3
Cxcl1
Cxcl3
Cxcl5
Ppbp
G-Csf: activation of granulocytes
PMN chemotaxis
PMN chemotaxis
PMN chemotaxis
CXCL-7: PMN chemotaxis
Ccl2
MCP-1: Monocyte chemotaxis, differentiation
Ccl3
Ccl4
Ccl7
MIP-1a: Monocyte chemotaxis, macrophage
diff.
MIP-1b: Monocyte chemotaxis, macrophage
diff.
MCP-3: Monocyte chemotaxis, macrophage
diff.
Injured infected/injured
uninfected fold change
1.25
-1.59
2.50
1.58
-3.23
1.26
1.59
1.26
1.25
Ltb
TNFb: Lymphocyte development in periphery
1.99
Cxcl10
Ccl17
Cxcl13
Th1, CD8, NK cell movement
Th2, Treg trafficking
B cell movement
6.35
5.05
-2.56
Role of cytokines in wound
healing in uninfected WOUND
Epidermis
Blood clot
Dermis
Capillaries
Damaged keratinocytes – release IL-1; and compared with UNINJURED tissue, this
Stimulates intact keratinocytes to express:
Mcp1 (↑12.6), Cxcl1 (↑43.9), Cxcl12 (↑2.5), Cxcl20 (↑63.7)
Recruits neutrophils, which express: Il1 (↑2.5), Il6 (↑33.7), Tnfa (↑2.5)
Recruits macrophages, which express: Spp1 (↑32), Il1, Il6, Tnfa
Regenerating keratinocytes express: Bmp2 (↑1.5), Bmp4 (↑2), Bmp6 (↑1.6), Bmp7 (↑2.5)
Effect of S. aureus infection on expression
of cytokines in injured tissue
Epidermis
Blood clot
S. aureus
Dermis
Capillaries
Damaged keratinocytes – release IL-1, BUT, compared to uninfected wound expression of
all cytokines expressed in uninfected is depressed or not enhanced
Keratinocyte expression of: Mcp1 (1.3), Cxcl1 (−1.6), Cxcl12 (−2), Cxcl20 (−1)
Neutrophil expression of: Il1 (−1.6), Il6 (-8.3), Tnfa (-2.6)
Macrophage expression of: Spp1 (-4.0), Il1, Il6, Tnfa
Keratinocyte regeneration delayed (?): Bmp2 (−1.3), Bmp4 (1.6), Bmp6 (−1), Bmp7 (1.6)
Acknowledgements
• Committee members: • Dr. Abdul Hamood Lab
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Dr. Jeter
Dr. Colmer-Hamood
Dr. Hamood
Dr. Zak
• Next Science
• Dr. Matt Myantti
• Dr. Cassandra Kruczek
•
•
•
•
Nyarie Dzvova
Nithya Mudaliar
Adeet Amin
Sabrina Siddiqui
• Kyle Miller M.S.
• Dr. Kendra Rumbaugh Lab