CHAPTER 5: In vivo and in vitro effects of injectable hormonal contraceptives on
5.4 Results
5.4.3 Effect of exogenous hormones on mDC lineage activation
To evaluate whether exogenous addition of DMPA influenced monocyte and DC activation or cytokine production, whole blood from healthy female donors not using HCs were stimulated with LPS (TLR4) or CpG (TLR9) with exogenous addition of natural (progesterone) or synthetic hormones (DMPA) or cortisol at varying concentrations (1, 10
-0.6 -0.4 -0.2 0.0 0.2 0.4 IL-6
IL-10
MCP-1 IL-8
IP-10
MIP-1a IL-1b
TNF-a IL-7
IL-1a GM-CSF
MIP-1b
DMPA
-1.0 -0.5 0.0 0.5 NET-EN
β -coefficient
and 100 nM). Figure 5.4 and 5.5 shows the gating strategy used to evaluate cytokine production (IL-6 and TNF-a) and cellular activation (CD40 and CD86 expression), respectively. For this analysis, monocytes were defined as HLA-DR+ CD14+ cells; mDCs were defined as HLA-DR+CD14-CD11c+CD123- cells; and pDCs were defined as HLA- DR+CD14-CD11c-CD123+ cells.
While both LPS and CpG increased mDC activation, with higher MFI expression of CD40 on CD11c+ cells, DMPA-, progesterone- and cortisol-treatment did not influence the level of mDC activation induced by these TLR agonists (Figure 5.6). In contrast to CD40, CD86 expression on mDCs was not increased by LPS or CpG stimulation and not affected by exogenously added hormones or cortisol (Figure 5.7). Similar analysis of CD14+ monocytes and CD123+ pDCs yielded the same results and have not been shown.
Figure 5.4 Gating strategy used to identify DC subsets and cytokine production of TNF-α and IL-6 that were either unstimulated (control), stimulated with LPS/CpG or a combination of LPS/CpG and hormone (DMPA, cortisol and progesterone). TNF-α production was defined by CD14- CD11c+ cells while IL-6 was defined by CD14- CD11c+ cells following stimulation of PBMCs with either CpG or LPS for 6 hours.
LYMPHOCYTES SINGLETS LEUKOCYTES CD14 AND HLA-DR POPULATION
CD11c and CD123 POPULATION TNF-α (UNSTIMULATED) TNF-α (CpG Stimulation)
IL-6 (UNSTIMULATED) IL-6 (CpG stimulation) IL-6 (CpG + DMPA stimulation) TNF-α (CpG + DMPA Stimulation)
0 50K 100K 150K 200K 250K
FSC-A 0
50K 100K 150K 200K 250K
SSC-A
64.2
0 50K 100K 150K 200K 250K
FSC-A 0
50K 100K 150K 200K 250K
FSC-H
84.3
0 103 104 105
<Alexa Fluor 700-A>: HLA-DR 0
102 103 104 105
<PE-A>: CD66ace
34.3
0 103 104 105
<Alexa Fluor 700-A>: HLA-DR 0
102 103 104 105
<Qdot 655-A>: CD14 11.3
12.6
0 102 103 104 105
<PerCP-Cy5-5-A>: CD123 0
102 103 104 105
<APC-Cy7-A>: CD11c 67
0.939
0 102 103 104 105
<PE-Cy7-A>: TNF-a 0
102 103 104 105
<PE-A>: CD66ace
22.3
0 102 103 104 105
<PE-Cy7-A>: TNF-a 0
102 103 104 105
<PE-A>: CD66ace
0.127
0 102 103 104 105
<PE-Cy7-A>: TNF-a 0
102 103 104 105
<PE-A>: CD66ace
2.62
0 102 103 104 105
<APC-A>: IL-6 0
102 103 104 105
<Qdot 605-A>: CD14
0.249
0 102 103 104 105
<APC-A>: IL-6 0
102 103 104 105
<Qdot 605-A>: CD14
18.6
0102 103 104 105
<APC-A>: IL-6 0
102 103 104 105
<Qdot 605-A>: CD14
7.98
Figure 5.5 Gating strategy for analysis of DC subset activation (CD86 and CD40). Whole
blood was either unstimulated (control), or stimulated with LPS/CpG or a combination of LPS/CpG and hormone (DMPA, cortisol and progesterone). Activation markers were defined as CD86 and CD40 following stimulation of PBMCs with either CpG or LPS.
LYMPHOCYTES SINGLETS LEUKOCYTES CD14 (monocytes) and HLA-DR populations
CD11c (mDCs) and CD123 (pDCs) CD40 expression (mDC activation marker)
CD86 expression (mDC/macrophage activation marker)
COUNT
CD40 Expression
COUNT
CD86 Expression Unstimulated Hormonal Contraception (HC)
A: Cortisol B: DMPA C: Progesterone HC + Ligand
CpG (TLR-9)
LPS (TLR-4)
Unstimulated Hormonal Contraception (HC)
A: Cortisol B: DMPA C: Progesterone HC + Ligand
CpG (TLR-9)
LPS (TLR-4)
A B C
A B C
0 50K 100K 150K 200K 250K
FSC-A 0
50K 100K 150K 200K 250K
SSC-A
64.2
0 50K 100K 150K 200K 250K
FSC-A 0
50K 100K 150K 200K 250K
FSC-H
84.3
0 103 104 105
<Alexa Fluor 700-A>: HLA-DR 0
102 103 104 105
<PE-A>: CD66ace
34.3
0 103 104 105
<Alexa Fluor 700-A>: HLA-DR 0
102 103 104 105
<Qdot 655-A>: CD14 11.3
12.6
0 102 103 104 105
<PerCP-Cy5-5-A>: CD123 0
102 103 104 105
<APC-Cy7-A>: CD11c 67
0.939
Figure 5.6 Effect of DMPA, cortisol and progesterone on CD40 expression (MFI) by mDCs stimulated with LPS (top panels) or CpG (bottom panels). Whole blood was either not stimulated (black bars), or stimulated with LPS/CpG (red bars), in the presence of DMPA (purple bars), cortisol (brown bars) or progesterone (blue bars). DMPA, progesterone and cortisol were used at varying concentrations (1, 10 and 100 nM). Box plots show the median, 25th and 75th percentile values, while whiskers show the 90 and 10% ranges; significance was determined using the Mann-Whitney test for non-parametric data. P-values <0.05 were considered significant.
Unstimulated LPS DMPA+LPS
Cortisol+LPS Progesterone + LPS 10
100
100nM
Unstimulated CpG DMPA+CpG
Cortisol+CpG Progesterone + CpG 10
100
100nM Unstimulated LPS
DMPA+LPS Cortisol+LPS
Progesterone + LPS 10
100
10nM
Unstimulated CpG
DMPA+CpG Cortisol+CpG
Progesterone + CpG 10
100
10nM Unstimulated LPS
DMPA+LPS Cortisol+LPS
Progesterone + LPS 10
100
1 nM
Unstimulated CpG DMPA+CpG
Cortisol+CpG Progesterone + CpG 10
100
1nM
CD40 Median fluorescent intensity (MFI)
Figure 5.7 Effect of DMPA, cortisol and progesterone on CD86 expression (MFI) by mDCs stimulated with LPS (top panels) or CpG (bottom panels). Whole blood was either not stimulated (black bars), or stimulated with LPS/CpG (red bars), in the presence of DMPA (purple bars), cortisol (brown bars) or progesterone (blue bars). DMPA, progesterone and cortisol were used at varying concentrations (1, 10 and 100 nM). Box plots show the median, 25th and 75th percentile values, while whiskers show the 90 and 10% ranges; significance was determined using the Mann-Whitney test for non-parametric data. P-values <0.05 were considered significant.
Unstimulated LPS DM
PA+LPS Cortisol+LPS
Progesterone + LPS 10
100
100nM
Unstimulated CpG DMPA+CpG
Cortisol+CpG Progesterone + CpG 10
100
100nM Unstimulated LPS
DMPA+LPS Cortisol+LPS
Progesterone + LPS 10
100
10nM
Unstimulated CpG
DMPA+CpG Cortisol+CpG
Progesterone + CpG 10
100
10nM Unstimulated LPS
DMPA+LPS Cortisol+LPS
Progesterone + LPS 10
100
1 nM
Unstimulated CpG
DMPA+CpG Cortisol+CpG
Progesterone + CpG 10
100
1nM
CD86 Median fluorescent intensity (MFI)