probiotics as novel · Diabetic foot ulcer Epidermolysis Bullosa Can probiotic extracts help to ......
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6/21/2014 1 Topical probiotics as novel treatments for skin in health and Disease Cath O’Neill Dermatological Sciences Research Group Faculty of Human and Medical Sciences The University of Manchester, UK ISAPP 2014 Skin Barrier Function is Critical to Health Epidermis Dermis subcutis The epidermis is a barrier that protects from: • dehydration • UVR • invasion by pathogens • Chemical pollutants • etc
Transcript of probiotics as novel · Diabetic foot ulcer Epidermolysis Bullosa Can probiotic extracts help to ......
6/21/2014
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Topical probiotics as novel treatments for skin in health and Disease
Cath O’Neill
Dermatological Sciences Research Group
Faculty of Human and Medical Sciences
The University of Manchester, UK
ISAPP 2014
Skin Barrier Function is Critical to Health
Epidermis
Dermis
subcutis
The epidermis is a barrier that protects from:
• dehydration • UVR • invasion by
pathogens• Chemical pollutants• etc
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Aberant Barrier function is evident in a number of skin diseases
Atopic dermatitis
Psoriasis
Diabetic foot ulcer
EpidermolysisBullosa
Can probiotic extracts help to repair/enhance/maintain the skin
barrier?
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Lysates of Specific Species increase the Rate of Re‐epithelialisation
0h 6h 12h 18h 24h
Con
LGG
LR
LF
LP
Lysates from L. rhamnosus GG stimulate keratinocyte migration and
proliferation
0
50
100
150
200
250
300
mig
rate
d c
ells
(%
)
* *
0
100
200
300
400
500
rate
of p
rolif
era
tion
(% c
ontr
ol)
* *
2h 4h 6h 8h 6h 12 18 24
control
treated
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Testing the lysate in human skin
Excision wound
LGG lysate promotes re‐epithelialisation in human skin
0
5
10
15
20
25
leng
th o
f to
ngu
e (
uM)
*
**
CONLGG
D0 D1 D3 D7
D0 D1 D3 D7
C
LGG
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LGG increases keratinocyte migration and proliferation
CON (D7) LGG (D7) CON (D7) LGG (D7)
cortactin Ki67
Microarray Analysis of the Effects of LGG on the Wound Healing Response
Function Number of genes
Cell adhesion 25
Apoptosis 163
viability 37
Cell Signalling 170
Chemotaxis 16
Differentiation 65
Protein expression 16
Response to infection 11
Inflammatory response 14
Cell migration 100
Cell proliferation 244
Cytostasis 10
Activation of Antigen presenting cells
11
P=<0.05
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Candidate genes were validated using qPCR
Gene Fold change
CXCL 54
CXCLR2 52
KGF 50
KGFR 43
LGG increases the protein levels of KGFR and CXCLR2
0
1
2
3
4
5
6
7
8
fold
cha
nge
Con LGG
β‐actin
CXCLR2
KGFR
CXCLR2 KGFR
*
*
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LGG increases the production of KGF and CXCL2
12 18 24 day 3 day 70
1
2
3
4
5
6
7
8
CX
CL
2 (u
g/m
l)
Time
* **
12 18 24 day 3 day 70
2
4
6
8
10
12
14
16
KG
F (
ug/m
l)
Time
*
*
*
CON
LGG
Conclusions
• Lysates from probiotic bacteria increase re‐epithelialisation of model wounds
• Species dependent effects
• Mechanisms include induction of chemokine‐receptor pairs leading to increased keratinocyte proliferation and migration
Bacterial lysates have a huge capacity to alter skin cell physiology!!
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Infection and Barrier Dysfunction
Atopic dermatitis (Eczema)‐ increased susceptibility to S. aureus infection
Foot ulcer – commonly associated with S. aureus infection
Can probiotic extracts protect against pathogens?
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Probiotics protect Keratinocytesfrom S. aureus induced toxicity in a
strain dependent manner
0
20
40
60
80
100
% v
iabili
ty
Con SA LRE LRH LS LRE LRH LS+SA +SA +SA
**
Prince et al, 2012
18
0
20
40
60
80
% v
iabili
ty (
%)
0
20
40
60
80
100
% v
iabi
lity
L. reuteri protects keratinocytes only if added prior to infection with S. aureus
Con SA SA+LR 1h 2h 4hPRE
Con SA 1h 2h 4hpost
*
**
Prince et al, 2012
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L. rhamnosus GG can rescue keratinocytes from S. aureus‐induced toxicity.
0
20
40
60
80
100
% v
iabili
tyCon SA 2h 4h 6h 8h 10h 12h
*
*
0
20
40
60
80
100
% v
iabi
lity
**
Con SA 2h 4h
Pre Post
Mechanism?
Hypothesis – inhibition of pathogen adhesion
*
SA SA+ α5β1
Integrin receptor blockade reduces S. aureus adhesion to keratinocytes
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Factors that reduce adhesion of S. aureus to keratinocytes also reduce its
toxicity
0
20
40
60
80
100%
via
bili
ty
Con SA SA +α5β1
*
*
Prince et al, 2012
LGG inhibits binding of S. aureus to keratinocytes
con
treated
PRE POST
**
0
1
2
3
4
5
6
7
8
no. o
f adh
eren
t S. a
ureu
s (l
og C
FU
/ml)
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L. Reuteri inhibits adhesion of S. aureus to keratinocytes
0
1
2
3
4
5
6
no. o
f ad
here
nt b
acte
ria (
log
10cf
u/m
l)
0
2
4
6
no
. of a
dh
ere
nt b
act
eri
a (
log
10 c
fu/m
l)
Co pre post
**
SA L. salivarius+ SA
con
treated
Lactobacilli protect keratinocytes from S. aureus induced toxicity in species
dependent ways:
• Competitive exclusion
• Competitive displacement
• and others!
LGG
L. Reuteri works only by exclusion of S. aureus
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What are the molecules in LGG mediating its effects?
Re‐epithelialisation
Inhibition of pathogen adhesion
Tight junction function (Sultanna et al, 2013)
Identifying efficacious molecules in the LGG Lysate
B1 40 aceto LGG RC 001:10_UV1_280nm B1 40 aceto LGG RC 001:10_UV2_215nm B1 40 aceto LGG RC 001:10_Cond B1 40 aceto LGG RC 001:10_Conc B1 40 aceto LGG RC 001:10_Fractions B1 40 aceto LGG RC 001:10_Inject B1 40 aceto LGG RC 001:10_Logbook
0
500
1000
1500
2000
2500
3000
3500
mAU
0.0 10.0 20.0 30.0 40.0 50.0 min
F2 A4A7 A11 B1B4B7 B11C2 C7 C13 D4 D9 D15 E6 E12 F3F7F12 G3 G8 G14 H5 H11 I1I4I7I11 J1J5J9J14K4 K9 K15L5L9L14 M5 Waste
MS/MS analysis
Inhibition of pathogen adhesion
re‐epithelialisation
Two molecules identified so far – inhibition of pathogen adhesionTJ
function
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Summary
•Probiotic bacteria offer great potential for use on skin
•Species dependent effects and mechanisms
•Two‐pronged approach to identify mechanisms and efficacious molecules
•Possible applications in:Wound careBarrier related conditions e.g. AD Cosmetic preparationsAnti‐infectivesEtc.
Acknowledgements:
Collaborators: Dr Andrew McBain (Manchester)Prof Ralf Paus (Lubech)
Staff and students: Dr Tessa Prince
Mrs Walaa Saeed
Dr Rawshan Choudray
Miss Reshma Sultanna
Miss Maryam Moradi
Mrs Carol Ward
