MPA
Understanding the effect of sex hormones and Lactobacilli on genital epithelial cell func2ons Hormone Alone - TNF-a
Hormone Alone - TNF-a
1Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON. 2Department of Microbiology and Immunology, Western University, London, ON. *
TNF-a pg/mL
5000
5000
0
0
M P A
P 4
E 2
N
H
M P A
E 2
H
N
M P A
P 4
P 4
Treatments
150 100
IL-1b pg/mL
IL-1b pg/mL
M P PA Percent Pre-Treatment 4
IL-8 pg/mL
IL-1a pg/mL
Percent Pre-Treatment
IL-1a pg/mL
IL-8 pg/mL
Percent Pre-Treatment
Percent Pre-Treatment
0.8
0.8 0.6 0.4
0.2
0
0.0
0.0
H
N
M P A
P 4
E 2
pa
C
ris
R
G
C
R
-1
-1
4
tNu sH
A
C on Mtro Pl
s
tu
L.
C
TNF-a pg/mL
L.
*
*
1000
** 1000
1000
800
800
GM-CSF pg/mL
s
pa
cr
is
C
L.
0
*
200 150
N
M P A
P 4
N
H
E 2
Percent Pre-Treatment* - L.0Crisp
400 200
M P A
M P P A 4 Percent Pre-TreatmentIL-1a pg/mL
P E 4 2
tu
4
-1
GM-CSF pg/mL R
-1
R
pa
is
cr
Treatments
p
ris
4
-1
-1
L.
R
C
C
R
G
C
L.
R
C on tro l
p
ris
4
C
R
G
C
L.
-1
-1
C on tro l
p ris
4 -1
C
R
G
C on tro l
tu
s
4
-1
R
C
R
M P A N
H
E N 2 H
IL-1aMPpg/mL Percent Pre-Treatment M TNF-a pg/mL P A P 4 GM-CSF pg/mL P A 4
E P4 2
L.
L.
N E2 H
-1
C on tro l
pa
is
cr
MIP-1aG pg/mL
IL-1b pg/mL
tu
s
4
R
C
R
G
H
N -1 R G
M P A
P 4
E 2
H
N
M P A
RANTES pg/mL
M P A
P 4
E 2
H N
20
30 20
Kruskal- Wallis = NS 10 Bonferonni = * between media/HIV Freidman = * between media/HIV an 0
Treatment 10
Treatments
B) TNF-‐α
*
*
20
2000
**
10
M P A
50
P 4
N
H
E 2
0
Treatments
1000 500
H IV
-1
R C -1 4
G R -1
R
M ed ia
H IV +
-1 C R
+ G R -1
4
H IV
-1 R G
H IV
C -1 R 4 C -1 4 + H IV
0
0
M ed ia
*
1500
R
100
*
G
150
H IV
30
TNF-a pg/mL
RANTES pg/mL
40
M Percent Pre-Treatment of TER P A
P 4
E 2
N
H
RANTES pg/mL
M P A
P 4
E 2
H
N
A) TER
Hormone Alone - RANTES
40
M P A
Treatments
+
Hormone Alone - RANTES
P 4
H
E 2
0
N
N
Treatments
M P A
P 4
H
E 2
P 4
M P A
Treatments E 2
0
M P A N
N
H
E 2
Treatments
H
M P A P 4
P 4
E 2
H
N
M P A
P 4
E 2
N
H
GECs pre-‐treated with probio2c lactobacilli confer protec2on from HIV-‐1 mediated barrier Combined NH and P4 (TNF-a) disrup2on 0
H
0
RANTES pg/mL
G R -1
R
+
R
2000 Treatment
2000 Treatment 0
0
30
N
P 4
E 2
H
N
IL-1a pg/mL
C -1 R 4 C -1 4 + H IV
MH P IV A
4000
G
5000
P -1 4
0
Treatments
Treatments
Combined all hormones (TER)
• To date, we have standardized op2mal 2me points (6-‐12 hours) and inocula2on dose (100 CFU/cell) for * * * the addi2on of Lactobacillus species to primary genital epithelial cells ** • Preliminary Results show: • Probio2c strains of lactobacilli increase GEC barrier func2ons • Hormones do not alter barrier func2ons, however they do differen2ally regulate produc2on of pro-‐ inflammatory cytokines • E2 appears to have an2-‐inflammatory effects on primary GEC • Probio2c lactobacilli protect the epithelial barrier from HIV mediated disrup2on • In summary, the current model will provide insight into how factors in the genital microenvironment can affect epithelial barrier func2on and subsequent HIV-‐1 infec2on in the FGT 150
100
50
L.
C
ris
s tu
pa
ACKNOWLEDGEMENTS I would like to thank Dr. Kaushic and all the members of the Kaushic lab for their valuable input and assistance in this project. Dr. Kaushic is the recipient of an Applied HIV Research Chair Award from the OHTN. This study is funded by a CIHR HIV Mucosal Team grant, where Dr. Kaushic is the team leader.
H IV
R C -1 4
+
-1
C
R
+
4
H IV
-1
R
G R -1
G
H IV
0
M ed ia
Percent Pre-Treatment of TER
4 4 4
4 -1 C R
pa R tu R C-1s RC 4 RC-1 C-14 -14 4
ris
C
Treatments
H
MCP-1 pg/mL
M P A
P 4
MCP-1 pg/mL
E 2
M P A
P E 4 2
M P P ATNF-a pg/mL 4
N
H
M P A
M P A
P 4
E 2
M P AN
H
E N 2 H
IL-10 pg/mL
P 4
E 2
H
N
0
H E I 2V
M P A
H
E P4 2
N E2 H
4000
N
M P A
P 4
6000
2.5
IL-6 pg/mL
H
5000
N
M P A
P 4
N
E 2
0
H
1.0 0
200
200
0
M Ned H ia
200
1.5
400
*
Treatments * Treatments
200
Figure 3: Effect of probiotic Lactobacillus strains GR-1 and RC-14, 0
1000
MCP-1 pg/mL
*
TNF-a pg/mL IL-1a pg/mL
400
600
M P A
IL-1a pg/mL
2.5 2.0
H
800
Hormone Alone - IL-10
P 4
Treatment
600
E 2
H
IL1a - NB
N
P 4
M P A
0
P 4
Treatments
0
1000
N
P 4
M P A
*
E 2
1000
2000
IL-6Epg/mL 2
2000
E 2
2
TNF-a pg/mL
3000
Treatment
600
400
15000 genital epithelial cells were grown in the presence or absence of 15000 0 800 physiological concentrations of female sex hormones, E2 (10-9) and P4 2.0 2.0 TNFa - GR1 TNFa - L. crisp TNFa RC14 -7) or hormonal -9 Combined NH and P4 (TNF-a) 600 600 (10 contraceptive, 10000 10000 MPA (10 ) and exposed to Lactobacillus Treatments Treatments *** 1.5 3000 3000 Treatments 1.5 *** spp. (GR-1, RC-14) at *** a concentration of 100 CFU/cell for 12 hours. A) 400 400 1.0 *** 1.0 TER measurements were observed at time 0 and 12 hours post bacterial 5000 2000 2000 200 5000 200 0.5 exposure. TERs are expressed as percent pre-treatment of initial TER at 0 0.5 0 0 * * * * 1000 hours 1000 on the x-axis. A minimum of five replicates per experimental 0.0 0 2000 0.0 0 condition were included. B-C) Apical supernatants were collected and Treatments Treatments 0 Treatments assayed 0by Millipore multi-analyte kit for the following cytokines: IL-1α and * Treatments Treatments 1500 IL1a - GR1 IL1a - RC14 IL1a - L. crisp TNF-α, and are expressed as pg/mL of cytokine production. Two replicates Treatment Treatment Treatment 800 800 per experimental condition were included. Data was analyzed by KruskalHormone Alone - MCP-1 1000 Hormone Alone - MCP-1 600 600 * Hormone Alone IL-6 Hormone Alone - 15000 IL-6 Hormonewith Alone - RANTES Wallis- non-parametric analysis of variance Dunn’s test to correct for Ho 15000 ** 8000 40 8000 400 40 500 multiple400comparisons *p
Hormone Alone - TNF-a
1Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, ON. 2Department of Microbiology and Immunology, Western University, London, ON. *
TNF-a pg/mL
5000
5000
0
0
M P A
P 4
E 2
N
H
M P A
E 2
H
N
M P A
P 4
P 4
Treatments
150 100
IL-1b pg/mL
IL-1b pg/mL
M P PA Percent Pre-Treatment 4
IL-8 pg/mL
IL-1a pg/mL
Percent Pre-Treatment
IL-1a pg/mL
IL-8 pg/mL
Percent Pre-Treatment
Percent Pre-Treatment
0.8
0.8 0.6 0.4
0.2
0
0.0
0.0
H
N
M P A
P 4
E 2
pa
C
ris
R
G
C
R
-1
-1
4
tNu sH
A
C on Mtro Pl
s
tu
L.
C
TNF-a pg/mL
L.
*
*
1000
** 1000
1000
800
800
GM-CSF pg/mL
s
pa
cr
is
C
L.
0
*
200 150
N
M P A
P 4
N
H
E 2
Percent Pre-Treatment* - L.0Crisp
400 200
M P A
M P P A 4 Percent Pre-TreatmentIL-1a pg/mL
P E 4 2
tu
4
-1
GM-CSF pg/mL R
-1
R
pa
is
cr
Treatments
p
ris
4
-1
-1
L.
R
C
C
R
G
C
L.
R
C on tro l
p
ris
4
C
R
G
C
L.
-1
-1
C on tro l
p ris
4 -1
C
R
G
C on tro l
tu
s
4
-1
R
C
R
M P A N
H
E N 2 H
IL-1aMPpg/mL Percent Pre-Treatment M TNF-a pg/mL P A P 4 GM-CSF pg/mL P A 4
E P4 2
L.
L.
N E2 H
-1
C on tro l
pa
is
cr
MIP-1aG pg/mL
IL-1b pg/mL
tu
s
4
R
C
R
G
H
N -1 R G
M P A
P 4
E 2
H
N
M P A
RANTES pg/mL
M P A
P 4
E 2
H N
20
30 20
Kruskal- Wallis = NS 10 Bonferonni = * between media/HIV Freidman = * between media/HIV an 0
Treatment 10
Treatments
B) TNF-‐α
*
*
20
2000
**
10
M P A
50
P 4
N
H
E 2
0
Treatments
1000 500
H IV
-1
R C -1 4
G R -1
R
M ed ia
H IV +
-1 C R
+ G R -1
4
H IV
-1 R G
H IV
C -1 R 4 C -1 4 + H IV
0
0
M ed ia
*
1500
R
100
*
G
150
H IV
30
TNF-a pg/mL
RANTES pg/mL
40
M Percent Pre-Treatment of TER P A
P 4
E 2
N
H
RANTES pg/mL
M P A
P 4
E 2
H
N
A) TER
Hormone Alone - RANTES
40
M P A
Treatments
+
Hormone Alone - RANTES
P 4
H
E 2
0
N
N
Treatments
M P A
P 4
H
E 2
P 4
M P A
Treatments E 2
0
M P A N
N
H
E 2
Treatments
H
M P A P 4
P 4
E 2
H
N
M P A
P 4
E 2
N
H
GECs pre-‐treated with probio2c lactobacilli confer protec2on from HIV-‐1 mediated barrier Combined NH and P4 (TNF-a) disrup2on 0
H
0
RANTES pg/mL
G R -1
R
+
R
2000 Treatment
2000 Treatment 0
0
30
N
P 4
E 2
H
N
IL-1a pg/mL
C -1 R 4 C -1 4 + H IV
MH P IV A
4000
G
5000
P -1 4
0
Treatments
Treatments
Combined all hormones (TER)
• To date, we have standardized op2mal 2me points (6-‐12 hours) and inocula2on dose (100 CFU/cell) for * * * the addi2on of Lactobacillus species to primary genital epithelial cells ** • Preliminary Results show: • Probio2c strains of lactobacilli increase GEC barrier func2ons • Hormones do not alter barrier func2ons, however they do differen2ally regulate produc2on of pro-‐ inflammatory cytokines • E2 appears to have an2-‐inflammatory effects on primary GEC • Probio2c lactobacilli protect the epithelial barrier from HIV mediated disrup2on • In summary, the current model will provide insight into how factors in the genital microenvironment can affect epithelial barrier func2on and subsequent HIV-‐1 infec2on in the FGT 150
100
50
L.
C
ris
s tu
pa
ACKNOWLEDGEMENTS I would like to thank Dr. Kaushic and all the members of the Kaushic lab for their valuable input and assistance in this project. Dr. Kaushic is the recipient of an Applied HIV Research Chair Award from the OHTN. This study is funded by a CIHR HIV Mucosal Team grant, where Dr. Kaushic is the team leader.
H IV
R C -1 4
+
-1
C
R
+
4
H IV
-1
R
G R -1
G
H IV
0
M ed ia
Percent Pre-Treatment of TER
4 4 4
4 -1 C R
pa R tu R C-1s RC 4 RC-1 C-14 -14 4
ris
C
Treatments
H
MCP-1 pg/mL
M P A
P 4
MCP-1 pg/mL
E 2
M P A
P E 4 2
M P P ATNF-a pg/mL 4
N
H
M P A
M P A
P 4
E 2
M P AN
H
E N 2 H
IL-10 pg/mL
P 4
E 2
H
N
0
H E I 2V
M P A
H
E P4 2
N E2 H
4000
N
M P A
P 4
6000
2.5
IL-6 pg/mL
H
5000
N
M P A
P 4
N
E 2
0
H
1.0 0
200
200
0
M Ned H ia
200
1.5
400
*
Treatments * Treatments
200
Figure 3: Effect of probiotic Lactobacillus strains GR-1 and RC-14, 0
1000
MCP-1 pg/mL
*
TNF-a pg/mL IL-1a pg/mL
400
600
M P A
IL-1a pg/mL
2.5 2.0
H
800
Hormone Alone - IL-10
P 4
Treatment
600
E 2
H
IL1a - NB
N
P 4
M P A
0
P 4
Treatments
0
1000
N
P 4
M P A
*
E 2
1000
2000
IL-6Epg/mL 2
2000
E 2
2
TNF-a pg/mL
3000
Treatment
600
400
15000 genital epithelial cells were grown in the presence or absence of 15000 0 800 physiological concentrations of female sex hormones, E2 (10-9) and P4 2.0 2.0 TNFa - GR1 TNFa - L. crisp TNFa RC14 -7) or hormonal -9 Combined NH and P4 (TNF-a) 600 600 (10 contraceptive, 10000 10000 MPA (10 ) and exposed to Lactobacillus Treatments Treatments *** 1.5 3000 3000 Treatments 1.5 *** spp. (GR-1, RC-14) at *** a concentration of 100 CFU/cell for 12 hours. A) 400 400 1.0 *** 1.0 TER measurements were observed at time 0 and 12 hours post bacterial 5000 2000 2000 200 5000 200 0.5 exposure. TERs are expressed as percent pre-treatment of initial TER at 0 0.5 0 0 * * * * 1000 hours 1000 on the x-axis. A minimum of five replicates per experimental 0.0 0 2000 0.0 0 condition were included. B-C) Apical supernatants were collected and Treatments Treatments 0 Treatments assayed 0by Millipore multi-analyte kit for the following cytokines: IL-1α and * Treatments Treatments 1500 IL1a - GR1 IL1a - RC14 IL1a - L. crisp TNF-α, and are expressed as pg/mL of cytokine production. Two replicates Treatment Treatment Treatment 800 800 per experimental condition were included. Data was analyzed by KruskalHormone Alone - MCP-1 1000 Hormone Alone - MCP-1 600 600 * Hormone Alone IL-6 Hormone Alone - 15000 IL-6 Hormonewith Alone - RANTES Wallis- non-parametric analysis of variance Dunn’s test to correct for Ho 15000 ** 8000 40 8000 400 40 500 multiple400comparisons *p
