Infection and innate immunity in the female genital tract
Professor Martin Sheldon FRCVS
Reproductive Immunology
ROME September 2016
Farmers and veterinarians are rising to the grand challenge to feed the world
1945 2015
But there is a problem: uterine infections increasingly cause endemic disease and reduce productivity
Bacterial infection of the uterus after parturition affects all dairy cattle
40% develop clinical disease
Animals remain infertile even after successful treatment of the disease
€1.4 billion/year in EU $0.6 billion/year in USA
Bacteria cause uterine disease
Widely accepted pathogens Griffin et al 1974 Therio 1, 91 Dohmen et al 1995 Therio 43, 1379
Sheldon et al 2002 Reproduction 123, 837 Wagener et al 2015 Vet Micro 175, 286
Gram-negative Escherichia coli
Gram-positive Trueperella pyogenes
Anaerobes Prevotella spp
Fusobacterium spp
Complex uterine microbiome
Jeon et al 2015 Appl Environ Microbiol 81:6324
4
Fertility
Normal
Sub-fertile
Infertile
Sheldon et al 2009 Biol Reprod 81:1025
Post partum uterine bacterial infection and disease is common in dairy cattle
0 10 20 30 40 50 60 700
20
40
60
80
100
Infection
Perc
enta
ge o
f ani
mal
s
Days post partum
Endometritis
Normal endometrium
Metritis
Subclinical endometritis
David Noakes
The mechanisms of inflammation and defence in the bovine endometrium - a missing component?
1. What evidence is there for the role of innate immunity in the postpartum uterus?
2. What factors might scale the inflammatory response in the endometrium?
3. What impact does uterine infection have on ovarian function?
Jules Hoffmann Bruce Beutler
Nobel Prize in Physiology or Medicine 2011 awarded for discoveries concerning Toll and Toll-like receptors (TLRs) for the activation of innate immunity by pathogen-associated molecular patterns
Innate immunity depends on cellular receptors that bind pathogen-associated molecular patterns
Macrophage Neutrophil
Cytokines IL-1β IL-6 IL-10 Chemokines IL-8 CCL5 Prostaglandins PLA2 PGE2 Antimicrobial peptides LAP TAP
TLR4
LPS E. coli
Attract and activate more macrophages
and neutrophils
Pathogen clearance
Hematopoietic innate immune cells sense pathogen-associated molecular patterns
NFκB ERK p38
CD14 MD2
TLR4
8
Inflammatory gene expression
Bacterial infection of the uterus causes disease with inflammation and damage of the endometrium
Innate immunity TLR4
Prostaglandins PLA2
Cytokines IL1B IL6
Chemokines IL8 CCL5
Antimicrobial peptides
Clinical disease Histopathology Gene transcription
Inflammation - neutrophils - macrophages - damage
Evidence for innate immune responses - in vivo acute phase protein response
4 8 1 2 1 6 2 0 2 4 2 8
0 .2
0 .4
0 .6
0 .8
D a y s p o s tp a r tu m
Ha
pto
glo
bin
(m
g/m
l)
0
N o rm a l n = 7
T o x ic m e tr it is n = 1 3
M e tr it is n = 1 3
Sheldon & Dobson 2001 Vet Rec 148, 172-175
Uterine infection increases the concentrations of LPS, acute phase proteins, and prostaglandin E in blood
Herath et al 2009 Endocrinology 150:1912-1920
E. coli
Haptoglobin
Prostaglandin F
LPS
α1-acid glycoprotein
Prostaglandin E
IL-6 IL-10 IL-1β
TNFα TLR4 IL-1α
©2009 Herath et al Reprod Biol Endo
Innate immunity in the postpartum endometrium
Herath et al 2009 Reprod Biol Endocrinol 7: 55
Endometrial organ cultures accumulate inflammatory mediators in response to live E. coli and T. pyogenes
Borges et al 2012 AJRI 67, 526
IL-1β IL-6 IL-8
Endometrial cells mount inflammatory response to pathogen-associated molecules
IL-6 IL-8 Epithelium
Stroma
IL-6
(p
g/m
l)
0
6 0
1 2 0
1 8 0
2 4 0
* **
*
*
**
**
*
*
IL-6
(p
g/m
l)
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0 **
*
*
*
IL-8
(p
g/m
l)
0
2 0
4 0
6 0
8 0
*
*
**
**
* *
IL-8
(p
g/m
l)0
3 0
6 0
9 0
1 2 0
*
*
*
* **
PAM
PAM
PAM
PAM
FSL-1
FSL-1
FSL-1
FSL-1
LPS
LPS
LPS
LPS
Turner et al 2014 Endocrinology 155, 1453
Endometrial cells detect pathogen-associated molecules
NFκB
0 min 45 min
LPS
vehicle
Cronin et al 2012 Biol Reprod 86, 51
Endometrial cells mount inflammatory response to pathogen-associated molecules via TLRs
TLR4 TLR2 TLR1 TLR6
Cronin et al 2012 Biol Reprod 86: 51 Turner et al 2014 Endocrinology 155, 1453-1465
si = siRNA
Primary stromal cells
LPS switches prostaglandin secretion in endometrial epithelial cells
Stromal cells Epithelial cells
0
50
100
150
200
*
*
Pros
tagl
andi
n (n
g/m
l) Prostaglandin E2
Prostaglandin F2a
Oxytocin LPS 1 mg/ml Oxytocin LPS 1 mg/ml0
50
100
150
200
*
*
Herath et al. 2009 Endocrinology 150:1912-1920
Pathogen-associated molecular patterns
Summary 1: endometrial cells generate inflammatory responses to pathogen-associated molecules
Danger model
Polly Matzinger
What factors might scale the inflammatory response in the endometrium?
DAMPs Damage-associated molecular patterns
Healey et al 2014 Scientific Reports 4: 7060
Host tissues and cells respond to IL-1α but not prototypical DAMPs, HMGB1 or hyaluronan
HA – hyaluronan – extracellular matrix DAMP HMGB1 – nuclear protein – cellular DAMP IL-1α - intracellular cytokine DAMP LPS – pathogen-associated molecule
Endometrium Blood
Healey et al 2014 Scientific Reports 4: 7060
Healy et al Scientific Reports 2014; 4: 7060
Endometrial cells sense and react to tissue damage during infection via interleukin 1α
Stroma
Epithelium
Supernatant
Supernatant
IL-1α scales endometritis when there is infection followed by cell damage
Healey et al 2014 Scientific Reports 4: 7060
Stroma
Epithelium
Countering infections requires a robust immune response and then resolution of the inflammation
What goes wrong in dairy cattle?
Inflammation
Days post partum
Chronic inflammation
Healthy
0 7 14 21 28
Reducing the availability of glucose blunts the innate immune response
Turner et al 2016 PLoS ONE 11: e0151416
0
2
4
6
8
IL-1
b (
pg
/mg
)
P < 0 .0 0 1
IL -1 b
0
5
1 0
1 5
2 0
IL-8
(p
g/m
g)
P < 0 .0 0 1
IL -8 C o n tro l
L P S
0
2 0 0
4 0 0
6 0 0
IL-6
(p
g/m
g)
P < 0 .0 0 1
IL -6
Endometrial organ culture
The sensor of cellular energy - AMPK - regulates the innate immune response
Ve
hic
le
AIC
AR
Co
mp
ou
nd
C
p h o s p o A M P K
to ta l A M P K
A IC A R
C o m p o u n d C
0 0 1 0 2 5 5 00
5
1 0
1 5
C o m p o u n d C (m M )
IL-1
b (
pg
/mg
)
P < 0 .0 0 1
0 0 1 0 2 5 5 00
1 0 0
2 0 0
3 0 0
C o m p o u n d C (m M )
IL-6
(p
g/m
g)
P < 0 .0 0 1
0 0 1 0 2 5 5 00
5
1 0
1 5
C o m p o u n d C (m M )
IL-8
(p
g/m
g)
P < 0 .0 0 1
0 0 2 5 0 5 0 0 1 0 0 00
5
1 0
1 5
A IC A R (m M )
IL-1
b (
pg
/mg
)
0 0 2 5 0 5 0 0 1 0 0 00
2 0 0
4 0 0
6 0 0
A IC A R (m M )
IL-6
(p
g/m
g)
P < 0 .0 0 1
0 0 2 5 0 5 0 0 1 0 0 00
1 0
2 0
3 0
4 0
A IC A R (m M )
IL-8
(p
g/m
g)
P < 0 .0 0 1
C o n tro l
L P S
Turner et al 2016 PLoS ONE 11: e0151416
Endometrial organ culture
Active resolution of inflammation
Phagocytosis and elimination of microbes
Sensing of vita-PAMPs
Inflammatory responses to
PAMPs
Sensing DAMPs from cell and tissue damage
Cell and tissue resilience counter
infections
Microbial virulence factors disrupt cells
Autocrine and paracrine regulation of innate immune response
Duration of microbial infection
Am
plitu
de o
f inf
lam
mat
ory
resp
onse
Nutrition and metabolism Ovarian hormones
Summary 2: The innate immune response reflects the severity of challenge and impact of the environment
Sheldon et al 2016 J Reprod Immunol
Sheldon et al 2002 Reproduction 123, 837 Herath et al 2007 Reproduction 134, 683
None Mild Moderate Severe
1
10
100
Folli
cual
r Flu
id L
PS (
ng/m
l)
Uterine inflammation
198
24
n = 7LPS
6 8 10 12 14 160
4
8
12
16
**
****
****
**** **
Folli
cle
diam
eter
(mm
) Normal uterus Uterine infection
6 8 10 12 14 160
1
2
3**
Oes
tradi
ol (p
g/m
l)
Days post partum
What impact does uterine infection have on ovarian function?
Conclusion: innate immunity is a key mechanism linking infection and infertility in the uterus and ovary
Sheldon IM, Price JC, Turner ML, Bromfield JJ, Cronin JJ 2014 Uterine infection and immunity in cattle. In “Reproduction in DomesticRuminants VIII”, pp 415-430. Editors: Juengel JJ, Miyamoto A, Price C, Reynolds LP, Smith MF, Webb R. Published by Context Products Ltd, Ashby de la Zouch, UK.