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Department of Physics, Chemistry and Biology

Master Thesis

In Vitro Study of Recruitment Ability of

Macrophages and Trophoblasts in Early Human

Pregnancy

Caroline Wendel

LiTH-IFM-A-Ex--2313--SE

Supervisor: Jan Ernerudh, Judit Svensson and Sofia Freland Linköpings

universitet

Examiner: Johan Edqvist, Linköpings universitet

Department of Physics, Chemistry and Biology Linköpings universitet

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Contents

1 Abstract ... 1

2 List of abbreviations ... 1

3 Introduction ... 1

4 Materials and Methods ... 3

4.1 Cells ... 4

4.1.1 Isolation of peripheral blood mononuclear cells (PBMC) from whole blood... 4

4.1.2 Isolation of monocytes from PBMC with positive CD14+ selection... 4

4.1.3 Differentiation of monocytes ... 4

4.1.4 HTR-8 Trophoblast cell line ... 5

4.1.5 CFSE labelling ... 5

4.2 Transwell system... 5

4.2.1 Transwell set-up ... 5

4.2.2 Matrigel ... 6

4.2.3 Chemokines ... 6

4.2.4 Cell type and concentration ... 6

4.3 Flow cytometry ... 7

4.3.1 Number of migrated cells ... 7

4.3.2 Extracellular staining with antibodies ... 7

4.3.3 Gating strategy ... 7

4.4 Statistics ... 8

5 Results ... 9

5.1 Methodological development ... 9

5.1.1 Migration of PBMC through uncoated transwell inserts ... 9

5.1.2 Migration of PBMC through various Matrigel concentrations ... 9

5.1.3 CFSE as a method to distinguish migrated PBMC from cell attractants ... 9

5.1.4 Alternatively activated macrophages as cell attractants ... 10

5.1.5 Chemokine mix (CCL2 and CXCL10) as cell attractant ... 11

5.1.6 Classically activated macrophages as cell attractants ... 12

5.1.7 CXCL12 as a cell attractant ... 12

5.1.8 Matrigel 1:5 and classically activated macrophages as cell attractants ... 13

5.1.9 Matrigel 1:2 and CXCL12 as cell attractant... 14

5.1.10 Matrigel 1:5 and CXCL12 as a cell attractant ... 15

5.2 Experiments ... 15

5.2.1 Classically activated macrophages ... 15

5.2.2 Alternatively activated macrophages ... 19

5.2.3 Trophoblast cell line ... 22

6 Discussion ... 25

7 Acknowledgements ... 28

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1 Abstract

The tolerance towards the semi-allogenic foetus is obtained through both systemic and local changes in the maternal immune response. Locally, in the decidua, the cell composition differs from that found in the blood; natural killer (NK) cells and macrophages being the major cell types. Decidual macrophages (dMØ), which are alternatively activated, and trophoblasts, placental cells of foetal origin, are believed to participate in the foetal tolerance at the foetal-maternal interface. To test the recruitment ability of macrophages and trophoblasts, and to test if these cells are responsible for the special cell composition in the decidua, a migration assay was established. In this migration assay peripheral blood mononuclear cells (PBMC) were allowed to migrate through Matrigel-coated transwell inserts into lower wells containing a recruiting stimulus. After testing several conditions, a protocol was established for further use. The results showed that in vitro alternatively activated macrophages, which display many of the surface markers as dMØ, hold a recruiting ability and recruit monocytes. Further there was an indication that trophoblasts also hold a recruiting ability. Neither cell types were shown to recruit NK cells. In conclusion, this study presents a suitable protocol for assessing chemotactic factors and different cell type’s ability to recruit cells from blood. Although the experiments need to be repeated and extended and the recruitment ability of dMØ needs to be evaluated in detail before a final conclusion can be drawn, the preliminary data indicated that macrophages and trophoblasts can recruit monocytes.

Keywords: Decidua, macrophages, Matrigel, migration assay, pregnancy, trophoblasts.

2 List of abbreviations

CFSE: Carboxyfluorescein succinimidyl ester

DMSO: Dimetylsulfoxid dMØ: Decidual macrophages

dNK cells: decidual natural killer cells EDTA: Ethylenediaminetetraacetic acid FCS: Foetal calf serum

GM-CSF: Granulocyte-macrophage colony-stimulating factor

HBSS: Hank’s balanced salt solution HLA: Human leukocyte antigen HSA: Human serum albumin IL: Interleukin

IFN: Interferon

LPS: Lipopolysaccharides

MACS: Magnetic activated cell sorting

M-CSF: Macrophage colony-stimulating factor

MHC: Major histocompability complex NK cells: Natural killer cells

NKT cells: Natural killer T cells ON: Over night

PBMC: Peripheral blood mononuclear cell PBS: Phosphate buffered saline

PEST: Penicillin-streptomycin solution RPMI: Roswell park memorial institute RT: Room temperature

SEM: Standard error of the mean SD: Standard deviation

Tc cells: Cytotoxic T cells Th cells: T helper cells TNF: Tumour-necrosis factor Treg: Regulatory T cell

3 Introduction

The immune system discriminates between self and non-self, and recognition of a foreign antigen results in an immune response (Murphy et al., 2008). Pregnancy therefore represents a paradox since the female body during normal pregnancy tolerates the presence of a semi-allogenic foetus, i.e. it expresses both maternal and paternal genes. The exact mechanisms behind the alterations in the maternal immune system that allow implantation, maintenance and even the development of the foetus’ own immune system are only partially known and

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understood. However, it is known that during pregnancy, both systemic and local changes in immune regulation of the mother occur.

At a systemic level, the balance between the CD4+ T helper (Th) cells is important. These

cells can be divided into two main populations, Th1 and Th2, based on their cytokine production (Mosmann et al., 1986). Th1 or Th2 associated cytokines force the immune response towards cellular or humoral immunity respectively (Mincheva-Nilsson, 2006). During pregnancy the systemic Th1/Th2balance is thought to be shifted towards a humoral Th2 response, which is believed to be of great importance for foetal survival (Wegmann et al., 1993).

During pregnancy there are also local changes; the cellular composition in the decidua, the endometrium during pregnancy, differs from that in blood. Decidual natural killer (dNK) cells are the dominant cell type. In blood, natural killer (NK) cells (CD56+CD3-) comprise 5-15 % of the leukocytes, whereas the specialized population of dNK cells comprises ~70 % of the leukocytes in the decidua during early pregnancy (Renaud and Graham, 2008) and is believed to be involved in remodelling of decidual arteries, which are important for functional placentation and placental growth (Ashkar et al., 2000).

Macrophages (CD14+) comprise the second largest population in the decidua and represent 20-30 % of the decidual leukocytes and the number is relatively constant throughout gestation. Further, nearly no B cells (CD19+) and only a small amount of T cells (CD3+) are found in the decidua (Renaud and Graham, 2008). Classically activated macrophages are associated with increased antigen presentation, secretion of pro-inflammatory cytokines and cell-mediated immunity, whereas alternatively activated macrophages are associated with increased endocytic activity, tissue repair and humoral immunity (Gordon and Taylor, 2005). Gustafsson et al. (2008) showed that the gene expression profile of human first trimester decidual macrophages (dMØ) corresponded with that of alternatively activated macrophages (Gustafsson et al., 2008), including an up-regulation of genes involved in immune suppression and anti-inflammatory functions, along with genes involved in tissue remodelling and angiogenesis. dMØ are believed to be involved in foetal tolerance and development.

Another cell population believed to be important is regulatory T cells (Tregs, CD4dim CD25bright) which are found in great numbers in the decidua during early pregnancy (Sasaki et al., 2004). These cells are powerful suppressors of inflammatory immune responses and can suppress immune responses towards alloantigen. Tregs have been shown to suppress the proliferation of naïve T helper cells by cell-to-cell contact, suggesting that Tregs contribute to the maternal immune tolerance towards the semi-allogenic foetus (Sasaki et al., 2004).

There are also other mechanisms, not entirely dependent on immune cells, which are involved in the maternal tolerance towards the foetus, e.g. involving trophoblast cells. Trophoblast cells constitute the outer layer of the blastocyst and are the precursor cells of the placenta. The hormone human Chorionic Gonadotropin (hCG) produced by trophoblasts facilitates the implantation of the embryo into the endometrium. After implantation the trophoblasts are able to differentiate into different populations with various functions e.g. hormone production or remodelling of maternal spiral arteries in the uterus (Kliman, 1999). Human trophoblast major histocompability complex (MHC) expression differs from other cells in that they lack the expression of MHC class II antigens (Peyman and Hammond, 1992) and that trophoblasts lack the two classical MHC class I antigens human leukocyte antigen (HLA)-A and –B (King

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et al., 2000b). However, the trophoblasts express the classical MHC class I antigen HLA-C (King et al., 2000b) and the nonclassical MHC class I antigens HLAE (King et al., 2000a), -F (Ishitani et al., 2003) and –G (King et al., 2000b). While the absence of most classical MHC molecules protects the trophoblast cells from a direct immune response by T cells, recognition by NK cells as self is crucial to avoid the cytotoxic function of these (Kurago et al., 1995). HLA-G, which has low polymorphism, has been suggested to interact with NK cells and inhibit their cytotoxic function (Khalil-Daher et al., 1999).

Thus, the decidua is infiltrated by large number of leukocytes. Given that the decidual leukocyte composition is so different from that in blood, there has to be a selective process involved in the recruitment of leukocytes from the blood to the decidua. It is likely that trophoblasts play a role in this along with the recruited leukocytes themselves. This selective migration ought to play an important role in the initiation of foetal tolerance as well as foetal development; further, failure in this mechanism is a potential reason for pregnancy complications and failure. An in vitro model is a suitable method to further study the recruitment potential of different cell types, which could also have implications for understanding immune regulation in general.

The aim of this study was to develop a method and test the recruitment of cells by decidual macrophages and trophoblasts, to investigate if they are involved in generating the special composition of leukocytes seen in the decidua.

4 Materials and Methods

A migration assay was used to investigate the recruitment ability of macrophages and trophoblasts (Figure 1). The idea is that cells plated in the lower well release chemotactic cytokines, called chemokines, which can recruit other cells. Which cells that are recruited depend on the release of different kinds of chemokines secreted by the cells plated in the lower well.

Figure 1. Transwell set-up. Recruiting cells were added to the lower wells. The cells to be recruited (in this set-up PBMC were used) were added to the transwell inserts coated with Matrigel which allowed migration into the lower wells. The recruiting cells were labelled with CFSE to distinguish them from migrating cells, when the cells were analysed using flow cytometry.

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4.1 Cells

4.1.1 Isolation of peripheral blood mononuclear cells (PBMC) from whole blood

PBMC were isolated to be used as migrating cells and to obtain monocytes for differentiation into macrophages.

Blood from healthy voluntary donors was collected in sodium-heparin vacuette tubes. Hank’s balanced salt solution (HBSS; GIBCO-Invitrogen, Paisley, Scotland, UK) was added to dilute the blood and Lymphoprep (Axis-shield, Oslo, Norway) was thereafter added under the diluted blood. The PBMC were isolated by density gradient centrifugation at 400g without brake for 30 minutes at room temperature (RT) and retrieved from the interface. The cells were resuspended in HBSS and centrifuged at 400 g at 4 °C for another 10 minutes. The pellet was thereafter washed twice in HBSS. Finally the PBMC were diluted in appropriate medium or buffer.

4.1.2 Isolation of monocytes from PBMC with positive CD14+ selection

Monocytes were isolated from PBMC to be used for differentiation into macrophages.

PBMC were isolated as mentioned above, with the addition of PBMC filtration through a magnetic activated cell sorting (MACS) pre-separation filter (Miltenyi biotech, Bergisch Gladbach, Germany) before the last washing step. In this step the filter was pre-wetted with HBSS before the PBMC were added; filtered PBMC were then washed three times with HBSS. Subsequently the pellet was resuspended in MACS buffer consisting of phosphate buffered saline (PBS; Medicago, Uppsala, Sweden) supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, St. Louis, MO, USA) and 0.5 % foetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA) for MACS sorting. 20 µl CD14 MicroBeads per 107 cells (Miltenyi biotech, Bergisch Gladbach, Germany) were added and the suspension was incubated for 15 minutes in the dark at 4 °C. MACS buffer was added and the PBMC were centrifuged at 300 g at 4 °C for 10 minutes. The pellet was resuspended in MACS buffer and the monocytes were positively separated from the rest of the PBMC through magnetic separation using an MS MACS separation column (Miltenyi biotech, Bergisch Gladbach, Germany) which was first pre-wetted with MACS buffer. The MACS separation column was then washed three times with MACS buffer and the monocytes were then pushed out of the column with a piston. The monocytes were subsequently centrifuged at 500 g at 4 °C for 5 minutes and resuspended in culture medium consisting of Roswell Park Memorial Institute 1640 (RPMI 1640; GIBCO-Invitrogen, Paisley, Scotland, UK) supplemented with 10 % FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin (PEST; BioWhittaker, Essen, Germany) and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) (1 % PEST/L-glutamine).

4.1.3 Differentiation of monocytes

Monocytes were differentiated into alternatively or classically activated macrophages.

The monocytes were grown in 24-well plates (Costar, NYC, NY, USA), 106 monocytes in each well. For alternatively activated macrophages, macrophage colony-stimulating factor (M-CSF; Preprotech, Rocky Hill, NJ, USA) and interleukin-10 (IL-10; Preprotech, Rocky Hill, NJ, USA) diluted in RPMI supplemented with 10 % FCS and 1 % PEST/L-glutamine to a final concentration of 50 ng/ml were added.

For classically activated macrophages granulocyte-macrophage colony-stimulating factor (GM-CSF; Preprotech, Rocky Hill, NJ, USA), lipopolysaccharides (LPS; Sigma-Aldrich, St.

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Louis, MO, USA) and interferon- γ (IFN-γ; Preprotech, Rocky Hill, NJ, USA) diluted in RPMI supplemented with 10 % FCS and 1 % PEST/ L-glutamine to a final concentration of 5 ng/ml, 2 ng/ml (if nothing else is specified) and 20 ng/ml respectively were added.

The monocytes were incubated in 37 °C with 5 % CO2. The culture medium was exchanged

after 2 to 3 days and the monocytes were again incubated in 37 °C and 5 % CO2 until 5 days

had passed since the initial separation of CD14+ cells.

4.1.4 HTR-8 Trophoblast cell line

The immortalized first trimester trophoblast cell line HTR-8, with properties of invasive extravillous cytotrophoblasts (Graham et al., 1993), was used as recruiting cells in some of the experiments. The cell line was originally transfected with SV40 and selected on G-418 containing medium and originates from (Graham et al., 1993) and provided by S. Sharma, Brown University, Providence, RI, USA. The cell line was grown to about 80 % confluence. Nonadherent cells were discarded and adhered cells removed enzymatically by trypsin (Sigma-Aldrich, St. Louis, MO, USA).

Approximately 1x106 HTR-8 cells were plated in new 250-cm2 tissue culture flasks (BD Bioscience, Le Pont De Claix, France) with RPMI 1640 supplemented with 5 % FCS and 1 % PEST/L-glutamine and maintained in an incubator at 37 °C and 5 % CO2. Trophoblasts used

for migration were centrifuged at 120 g at RT for 5 minutes and labelled with CFSE as described below.

4.1.5 CFSE labelling

The cytosolic dye carboxyfluorescein succinimidyl ester (CFSE, Sigma-Aldrich, St. Louis, MO, USA) was used to label cells used as cell attractants. When analysing the cells by flow cytometry this labelling allowed to distinguish migrated cells from recruiting cells.

The differentiated macrophages were carefully scraped off and centrifuged at 500 g at 4 °C for 5 minutes; the pellet was resuspended in PBS containing 0.1 % FCS. 2.5 µl CFSE diluted in dimetylsulfoxid (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 0.25µM (if nothing else is specified) was added per 106 cells for internal labelling of the macrophages or trophoblasts, followed by incubation in 37 °C for 10 minutes. Ice cold RPMI 1640 supplemented with 10 % FCS and 1 % PEST/L-glutamine was added to block the reaction, followed by incubation on ice for 5 minutes. The macrophages or trophoblasts were centrifuged at 400 g at 4˚C for 10 minutes and washed twice in RPMI 1640 supplemented with 10 % FCS and 1 % PEST/L-glutamine followed by a last centrifugation at 400 g at 4˚C for 10 minutes. The final pellet was thereafter resuspended in RPMI 1640 supplemented with 0.5 % human serum albumin (HSA; Octapharma, Stockholm, Sweden) and 1 % PEST/ L-glutamine.

4.2 Transwell system 4.2.1 Transwell set-up

Culture plates (24-well; Costar, NY, USA) with transwell inserts with polycarbonate membranes with a pore size of 5µm (Costar, NY, USA) were used for these experiments. The transwell system is shown in Figure 1.

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4.2.2 Matrigel

Matrigel (growth factor reduced, BD Biosciences, Bedford, MA, USA) was used to provide more resistance to migrating cells and to make the migration assay more in-vivo like. The Matrigel was thawed on ice, mixed to homogeneity and diluted with RPMI supplemented with 0.5 % HSA and 1% PEST/L-glutamine to a final concentration of 1:1, 1:2, 1:5, 1:7.5, 1:10 and 1:20. The Matrigel was kept on ice and cold pipette tips were used. The concentration 1:5 was used if not otherwise specified.

The transwell inserts were coated with 100 µl of Matrigel (if nothing else is specified) and allowed to gelatinize for 1h at room temperature. The Matrigel was then carefully washed twice with RPMI supplemented with 0.5 % HSA and 1 % PEST/L-glutamine. The Matrigel-coated inserts were then incubated in the wells with macrophages, trophoblasts or chemokines for 1h in an incubator at 37 °C and 5 % CO2 before PBMC were added to the transwell

inserts.

4.2.3 Chemokines

Due to their known recruiting ability, chemokines were used as cell attractants to test the transwell system. 100 ng/ml CCL2, 10 ng/ml CXCL13, 10 ng/ml CCL17 and 50 ng/ml CXCL10 (Peprotech, Rocky Hill, NJ, USA) were used in the initial transwell experiment with uncoated transwell inserts. The types of cells attracted by the different chemokines are displayed in Table 1. The migration time was either 2 hours or overnight (ON). The migrated cells were examined using flow cytometry.

A mix of 100 ng/ml CCL2 and 50 ng/ml CXCL10 as well as CXCL12 (Peprotech, Rocky Hill, NJ, USA) in the concentrations 10, 50 or 250 ng/ml were used to assess selective migration compared to spontaneous migration through Matrigel-coated transwell inserts. All the chemokines were diluted in RPMI 1640 supplemented with 0.5 % HSA and 1 % PEST/L-glutamine to the final concentrations stated above.

Table 1. Example of leukocyte populations attracted by the chemokines used and the receptors specific for the chemokines, as reviewed by (Mantovani et al., 2004).

Chemokine Receptor Leukocyte

CCL2 CCR2 Monocytes, Natural killer cells, B cells etc.

CXCL10 CXCR3 Th1, Natural killer cells

CXCL12 CXCR4 Several

CXCL13 CXCR5 B cells

CCL17 CCR4 Th2, Natural killer cells, monocytes etc.

4.2.4 Cell type and concentration

100 µl cell suspension containing 5x105 or 1x106 PBMC was added to each transwell insert. 1.25x105, 2.5x105 or 5x105 classically or alternatively activated macrophages, 7.5x104, 1.25x105 or 2.5x105 trophoblasts or chemokine solution (all 500 µl) were added to the lower wells.

Medium alone was used as a negative control in all experiments and CXCL12 at a final concentration of 250 ng/ml was used as a positive control in the trophoblast experiments. In each experiment wells were used in triplicates.

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4.3 Flow cytometry

Flow cytometry was used to investigate the number and type of cells that had migrated into the lower wells.

4.3.1 Number of migrated cells

Cells that had migrated from the transwell inserts to the lower wells were harvested and analysed with a FACSCanto II using FACSDiva software (BD Biosciences, San Jose, CA, USA). Cells were collected for 60 seconds and the number of migrated cells was calculated using Trucount tubes (BD Biosciences, San Jose, CA, USA). In some of the experiments, migrated cells were harvested and 50 µl from each well was diluted in PBS supplemented with 0.1 % FCS for analysis of number of migrated cells. The remaining cells were used for extracellular staining.

4.3.2 Extracellular staining with antibodies

The cell composition of PBMC before and after migration was investigated through extracellular staining with antibodies.

PBMC (before and after migration) were centrifuged at 500 g at 4 °C for 5 minutes and resuspended in PBS supplemented with 0.1 % FCS and incubated with antibodies for 30 minutes at 4 °C in the dark. PBS supplemented with 0.1 % FCS was added, followed by centrifugation at 500 g at 4 °C for 5 minutes. The pellet was resuspended in PBS supplemented with 0.1 % FCS.

The antibodies and their concentrations are commonly used in the lab and titrated to work on FACSCanto II. The conjugated antibodies used are showed in Table 2.

Table 2 . Antibodies used for extracellular staining.

Antibody Fluorochrome Source

CD3 Pacific blue BD Biosciences, San Jose, CA, USA

CD3 PE BD Biosciences, San Jose, CA; USA

CD4 PerCP BD Biosciences, San Jose, CA, USA

CD8 V450 BD Biosciences, San Jose, CA, USA

CD14 PC7 Beckman Coulter, Brea, CA, USA

CD19 APC BD Biosciences, San Jose, CA, USA

CD25 APC BD Biosciences, San Jose, CA, USA

CD56 PE BD Biosciences, San Jose, CA, USA

CD56 V450 BD Biosciences, San Jose, CA, USA

4.3.3 Gating strategy

Flow cytometry was used to differentiate between different cell populations based on their forward and side scatter (representing size and granularity, respectively) along with expression of cell specific extracellular markers detected with fluorescent antibodies.

Migrated PBMC were identified as CFSE negative. T cells were analysed based on their forward and side scatter, and gated for expression of CD3. Th cells were gated as CD3+CD4+, cytotoxic T (Tc) cells as CD3+CD8+ and Natural killer T (NKT) cells as CD56+ CD3+. Tregs were gated as CD4dimCD25bright within the CD3 positive population (Mjosberg et al., 2009). NK cells were gated as CD56+CD3- and B cells as CD19+ within the lymphocyte population. Monocytes were also analysed based on their forward and side scatter, and gated for expression of CD14. Figure 2 shows how the different cell populations were gated (the boxes are the gates).

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Figure 2. Shows how the different cell populations were gated, A) CD3+( T cells), B) CD4+ (Th cells) and CD8+ (Tc cells) , C) CD14+ (Monocytes), D) CD4dimCD25bright (Treg) E) CD56+CD3+ (NKT cells) and CD56+CD3- (NK cells), F) CD19+ (B cells) .

4.4 Statistics

Statistical significance was assessed by using repeated measures One-way ANOVA and Dunnett’s multiple comparison test. A p-value of ≤ 0.05 was considered statistically significant. CD8+ CD4dimCD25bright 102 102 102 102 102 102 103 103 103 103 103 103 104 104 104 104 104 104 105 105 105 105 105 105 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 50 100 150 200 250 FSC CD3+ (T cells) CD4

CD4+ (Th cells) & CD8+ (Tc cells)

CD3

CD14+ (Monocytes)

CD4

CD4dimCD25bright (Treg cells)

CD3 CD56+CD3+ (NKT cells) & CD56+CD3- (NK cells) CD3 CD19+ (B cells) CD8 CD14 CD25 CD19 CD56 CD3 CD3+ CD4+ CD14+ CD56+CD3- CD56+CD3+ CD19+ A. C. D. F. E. B.

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5 Results

5.1 Methodological development

In the first part of the project a method was developed to allow in vitro investigation of the recruitment ability of decidual macrophages and trophoblasts. The principle of the method is to allow selective migration of PBMC through transwell inserts into the lower wells, where the macrophages or trophoblasts are situated.

5.1.1 Migration of PBMC through uncoated transwell inserts

Initially 1x106 and 5x105 PBMC were allowed to migrate ON and 1x106 PBMC were allowed to migrate 2 h through uncoated transwell inserts. The lower wells contained one of the chemokines CCL2, CXCL13, CCL17, CXCL10 or medium alone as a negative control. The PBMC composition before migration and in the lower wells after migration was investigated through labelling of cell surface markers followed by analysis by flow cytometry.

The expected difference in cell composition, due to the recruitment ability of the different chemokines in the lower wells, could not be seen regardless of migration time 2 h or ON (data not shown).

5.1.2 Migration of PBMC through various Matrigel concentrations

To increase the selectivity of the migration from the transwell insert into the lower well, a Matrigel layer was introduced. Different Matrigel concentrations were tested and 1x106 PBMC were allowed to migrate 2 h through Matrigel-coated transwell inserts. The Matrigel concentrations tried were 1:1, 1:5, 1:7.5, 1:10 and 1:20. The lower wells contained a chemokine mix of CCL2 and CXCL10. The amount of migrated PBMC was calculated using TruCount tubes and flow cytometry.

The experiment was performed twice (Test I and Test II). Due to great variations in the amount of PBMC that had migrated between Test I and II the data was found to be inconclusive (Table 3). The Matrigel concentration 1:7.5 was chosen for further studies based on that a sufficient amount of PBMC, for analysis, had migrated into the lower wells and also based on previous studies (Renaud et al., 2009).

Table 3.Ratio of migrated PBMC through transwell inserts coated with various Matrigel concentrations. Lower wells with chemokine as cell attractants were compared to control wells with medium alone (+/- equivalent to a ratio of ~1, + to a ratio of > 2.9, - to a ratio of < 0.5). Matrigel concentration 1:1 1:5 1:7.5 1:10 1:20 Test I +/- + +/- +/- - Test II +/- + +/- - +/-

5.1.3 CFSE as a method to distinguish migrated PBMC from cell attractants

The cytosolic dye CFSE was tested as a method to distinguish recruiting cells from migrated cells found in the lower wells. Alternatively activated macrophages were labelled with the cytosolic dye CFSE and mixed with PBMC. CFSE was found to be a sufficient method to distinguish differentiated macrophages from non-labelled PBMC. Differentiated macrophages could easily be defined as CFSE positive and PBMC as CFSE negative when using flow cytometry (Figure 3).

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The CFSE concentration of 0.5µM was reduced to 0.25µM for further experiments since the signal otherwise was too high and gave too much background to the test. CFSE is commonly used in proliferation studies but usually in higher concentrations (Tario et al., 2007).

Figure 3. Shows how CFSE negative cells (migrated PBMC) and CFSE positive (recruiting)

cells are distinguished from each other using flow cytometry.

5.1.4 Alternatively activated macrophages as cell attractants

Alternatively activated macrophages were thereafter tried as cell attractants. 1x106 PBMC were allowed to migrate 2 h through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:7.5 and the lower wells contained either medium alone or medium with 2.5x105 or 5x105 alternatively activated macrophages. This experiment was repeated once. Initially there was an indication of selective migration into wells containing macrophages compared with medium alone. Further, an increase in the number of migrated PBMC was seen with increased number of macrophages, i.e. a dose dependency (Figure 4A). The same selective migration could not be seen when repeating the experiment (Figure 4B), although there was a slight increase in migrated PBMC into wells that contained 5x105 macrophages.

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11 -250 0 00 M Ø 500 0 00 M Ø 0 25000 50000 75000 100000 125000 150000 175000 No . o f m ig ra te d P B M C -250 0 00 M Ø 500 0 00 M Ø 0 25000 50000 75000 100000 125000 150000 175000 No . o f m ig ra te d P B M C A B

Figure 4. 1x106 PBMC migrated 2 h through Matrigel-coated transwell inserts. The lower

wells contained either medium alone (the left bar in each panel) or medium with 2.5x105 or

5x105 alternatively activated macrophages. The Matrigel concentration used was 1:7.5.

Figure A shows an increase in number of migrated PBMC into macrophage containing wells; this increase appears to be dose-dependent. The experiment was repeated once (Figure 4B); the same dose-dependent increase in number of migrated macrophages was not as clear. The Figure shows the mean and SD of migrated PBMC, each well concentration was run in triplicates.

5.1.5 Chemokine mix (CCL2 and CXCL10) as cell attractant

A chemokine mix of CCL2 and CXCL10 was thereafter used as cell attractants in order to investigate if selective migration could be achieved. 1x106 PBMC were allowed to migrate for 2 h through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:7.5. More PBMC migrated into wells containing medium alone than into wells containing the chemokine mix (Figure 5).

-CCL2 & C XCL1 0 0 50000 100000 150000 200000 250000 N o . o f m ig ra te d P B M C

Figure 5. 1x106 PBMC migrated 2 h through Matrigel-coated transwell inserts.

The Matrigel concentration used was 1:7.5 and the lower wells contained either medium alone or a chemokine mix of CCL2 and CXCL10. The graph shows mean and SD of migrated PBMC, each well concentration was run in triplicates.

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5.1.6 Classically activated macrophages as cell attractants

1x106 PBMC migrated 2 h through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:7.5 and the lower wells contained either medium alone or medium with 2.5x105 or 5x105 classically activated macrophages (Figure 6).

No significant difference was found when comparing the amount of PBMC that had migrated into wells with medium alone to wells with medium containing different amounts of macrophages. -250 000 MØ 500 000MØ 0 25000 50000 75000 100000 125000 N o . o f mi g ra te d P B MC

Figure 6. 1x106 PBMC migrated 2 h through Matrigel-coated transwell inserts. The Matrigel

concentration used was 1:7.5 and the lower wells contained either medium alone or medium

with 2.5x105 or 5x105 classically activated macrophages. The graph shows mean and SD of

migrated PBMC, each well concentration was run in triplicates.

5.1.7 CXCL12 as a cell attractant

The recruiting ability of CXCL12, which was recommended as a positive control by M. Quiding-Järbrink (The Sahlgrenska Academy at University of Gothenburg, Sweden), was investigated in these experiments.

1x106 PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:7.5 and the lower wells contained CXCL12 at a final concentration of 10 ng/ml, 50 ng/ml, 250 ng/ml or medium alone (Figure 7). The experiment was repeated once.

The number of migrated PBMC in the various CXCL12 concentrations was dose-dependent in both experiment A and B although more apparent in the first experiment (Figure 7 A).

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13 - 10ng 50ng 250n g 0 25000 50000 75000 100000 N o . o f m ig ra te d P B M C -10n g 50n g 250 ng 0 25000 50000 75000 100000 N o . o f mi g ra te d P B MC A B

Figure 7. 1x106 PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel

concentration used was 1:7.5 and the lower wells contained either medium alone or medium with CXCL12 at a final concentration of 10 ng/ml, 50 ng/ml or 250ng /ml. A dose-dependent difference in migrated PBMC could be seen in both experiments (the first experiment (A) and also in the second experiment (B)), although more apparent in A. The graph shows mean and SD of migrated PBMC, each well concentration was run in triplicates.

5.1.8 Matrigel 1:5 and classically activated macrophages as cell attractants

For this experiment the Matrigel concentration was changed to a dilution of 1:5. This higher concentration was used in the hope that a thicker coating of the transwell inserts would present more resistance to migrating cells and thus reduce the spontaneous migration.

1x106 (Figure 8A) or 5x105 (Figure 8B) PBMC migrated ON through the Matrigel-coated transwell inserts. The lower wells contained either medium alone or medium with 2.5x105 or 5x105 classically activated macrophages (Figure 8).

More PBMC migrated into lower wells that contained medium alone than into wells containing classically activated macrophages. We could also conclude that the Matrigel concentration of 1:5 was not too thick to obtain enough PBMC for flow cytometry analysis.

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14 -250 0 00 M Ø 500 0 00 M Ø 0 20000 40000 60000 80000 100000 120000 140000 No . o f m ig ra te d P B M C -500 0 00 M Ø 0 20000 40000 60000 80000 100000 120000 140000 No . o f m ig ra te d P B M C A B

Figure 8. 1x106 (A) or 5x105 (B) PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:5 and the lower wells contained either

medium alone or medium with 2.5x105 or 5x105 classically activated macrophages. The graph

shows mean and SD of migrated PBMC, each well concentration was run in triplicates.

5.1.9 Matrigel 1:2 and CXCL12 as cell attractant

The Matrigel concentration was changed to 1:2 which gives a solid gel instead of a coating. This experiment was done to see which effect a gel instead of a coating would have on the spontaneous migration.

5x105 PBMC migrated 2 h (Figure 9 A) or ON (Figure 9 B) and the lower wells contained either medium alone or medium with CXCL12 to a final concentration of 50 ng/ml or 250ng /ml. The Matrigel concentration 1:2 was found to be too thick to allow a sufficient amount of PBMC to migrate through for analysis by flow cytometry.

-50ng 250n g 0 1000 2000 3000 N o . o f m ig ra te d P B M C -50ng 250ng 0 1000 2000 3000 No . o f m ig ra te d PB M C A B

Figure 9. 5x105 PBMC migrated 2 h (A) or ON (B) through the transwell inserts with a Matrigel concentration of 1:2. The lower wells contained either medium alone or medium with CXCL12 to a final concentration of 50 ng/ml or 250ng /ml. Very few cells migrated into the lower wells. The graphs show mean and range of migrated cells and each well concentration was run in triplicates.

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5.1.10 Matrigel 1:5 and CXCL12 as a cell attractant

The Matrigel concentration 1:5 was again used since the concentration 1:2 was shown to be too thick and did not allow a sufficient number of PBMC to migrate. CXCL12 at a final concentration of 10 ng/ml, 50 ng/ml or 250ng /ml or medium alone was used as a cell attractant. 5x105 PBMC were allowed to migrate ON through Matrigel-coated transwell inserts.

The difference in migrated PBMC into wells containing 250 ng/ml CXCL12 and the wells with medium alone was found to be significant (Figure 10).

-10ng /mL 50ng /mL 250n g/m L 0 10000 20000 30000 40000 50000 60000 70000 p< 0.01 No . o f m ig ra te d P B M C

Figure 10. 5x105 PBMC migrated ON through Matrigel-coated transwell inserts.

The Matrigel concentration used was 1:5 and the lower wells contained either medium alone or medium with CXCL12 with a final concentration of 10 ng/ml, 50 ng/ml or 250ng /ml. These parameters gave a significant (p<0.01) selective migration into wells that contained medium with 250 ng/ml CXCL12 as compared with medium alone (n=3). The graph shows mean and SEM of migrated cells and each well concentration was run in triplicates.

5.2 Experiments

In the second part of the project the established migration assay was used to investigate the recruiting ability of macrophages and trophoblasts.

5.2.1 Classically activated macrophages

These experiments investigated the recruiting ability of classically activated macrophages. 5x105 PBMC were allowed to migrate ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:5 and the lower wells contained either medium alone or medium with 1.25x105, 2.5x105 or 5x105 classically activated macrophages (Figure 11). A significant (p<0.05) difference was observed when comparing the amount of PBMC recruited by 1.25x105 classically activated macrophages as compared with medium alone. In one of the experiments, the composition of the migrated PBMC was analysed with flow cytometry (Figure 12 and 13). The results suggested that the monocytes are recruited to a

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much higher degree than other cells, both spontaneously (as seen in wells with medium alone) and actively by classically activated macrophages (Figure 12C and 13C). Interestingly, NK cells were also found in a higher percentage in the lower wells after migration compared to before migration, although there was no apparent difference between control wells and wells with macrophages (Figure 12D and 13D).

-125 000 MØ 250 000 MØ 500 000 MØ 0 10000 20000 30000 40000 50000 60000 70000 p< 0.05 N o . o f m ig ra te d P B M C

Figure 11. 5x105 PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:5 and the lower wells contained either medium alone or medium with 1.25x105, 2.50x105 or 5x105 classically activated macrophages. A significant (p<0.05) difference was observed when comparing the amount of PBMC recruited by 1.25x105 classically activated macrophages as compared with medium alone (n=3) .Each well concentration was run in triplicates. The graph shows mean and SEM.

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17 CD3+ (T cells) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f ly m p h o c y te s CD4+ (Th cells) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f ly m p h o c y te s CD14+ (Monocytes) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f P B M C CD56+ CD3- (NK cells) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f ly m p h o c y te s CD4+ CD25+ (Treg) Befo re -125 000 250 000 500 000 0 1 2 3 4 5 % o f ly m p h o c y te s CD56+ CD3+ (NKT cells) Befo re -125 000 250 000 500 000 0 1 2 3 4 5 % o f l y mp h o c y te s A B C D E F

Figure 12. Flow cytometry analysis of migrated PBMC.

The graphs show the percentage of A) CD3+(T cells), B) CD4+ (Th cells), C) CD14+ (Monocytes), D) CD56+CD3- (NK cells), E) CD4dimCD25bright (Treg) and F) CD56+CD3+ (NKT cells) before and after migration in each well (n=1) (note the different scale on y-axis).

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18 CD3+ (T cells) 125 000 MØ 250 000 MØ 500 000 MØ -10 -5 0 5 10 p e rc e n til e f o ld c h a n g e CD4+ (Th cells) 125 000 MØ 250 000 MØ 500 000 MØ -10 -5 0 5 10 p e rc e n til e f o ld c h a n g e CD14+ (Monocytes) 125 000 M Ø 250 000 M Ø 500 000 M Ø -10 0 10 20 30 p e rc e n til e f o ld c h a n g e CD56+ CD3- (NK cells) 125 000 MØ 250 000 MØ 500 000 MØ -10 -5 0 5 10 p e rc e n til e f o ld c h a n g e CD4+ CD25+ (Treg) 125 000 MØ 250 000 MØ 500 000 MØ -2 -1 0 1 2 p e rc e n til e f o ld c h a n g e CD56+ CD3+ (NKT cells) 125 000 MØ 250 000 MØ 500 000 MØ -2 -1 0 1 2 p e rc e n til e f o ld c h a n g e A B C D E F

Figure 13. Percentile fold change of migrated PBMC into wells containing 1.25x105, 2.5x105 or 5x105classically activated macrophages compared to wells containing medium alone. A) CD3+(T cells), B) CD4+ (Th cells), C) CD14+ (Monocytes), D) CD56+CD3- (NK cells), E) CD4dimCD25bright (Treg) and F) CD56+CD3+ (NKT cells) (n=1) (note the different scales on y-axis).

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5.2.2 Alternatively activated macrophages

These experiments investigated the recruiting ability of alternatively activated macrophages. 5x105 PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:5 and the lower wells contained either medium alone or medium with 1.25x105, 2.5x105 or 5x105 alternatively activated macrophages (Figure 14). M-CSF and IL-10 (50 ng/ml) were used to obtain the alternatively activated macrophage phenotype in contrast to previous experiments on alternatively activated macrophages where only M-CSF was used.

A significant (p<0.05) difference was observed when comparing the amount of PBMC recruited by 5x105 alternatively activated macrophages as compared with medium alone. The composition of PBMC was investigated using flow cytometry (Figure 15 and 16). The results showed that significantly more monocytes migrated into wells containing alternatively activated macrophages compared to medium alone (Figure 15C and 16C).

-125 000 250 000 500 000 0 10000 20000 30000 40000 50000 60000 70000 80000 p<0.05 N o . o f m ig ra te d PB M C

Figure 14. 5x105 PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:5 and the lower wells contained either medium alone or medium with 1.25x105, 2.5x105 or 5x105 alternatively activated macrophages (n=3).

A significant (p<0.05) difference was observed when comparing the amount of PBMC recruited by 5x105 alternatively activated macrophages as compared with medium alone (n=3). Each well concentration was run in triplicates. The graph shows mean and SEM.

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20 A B C D E F CD3+ (T cells) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f ly m p h o c y te s CD4+ (Th cells) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f ly m p h o c y te s CD14+ (Monocytes) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f PB M C CD56+ CD3- (NK cells) Bef ore -125 000 250 000 500 000 0 10 20 30 40 50 60 70 80 % o f ly m p h o c y te s CD4+ CD25+ (Treg) Bef ore -125 0 00 M Ø 250 0 00 M Ø 500 0 00 M Ø 0 1 2 3 4 5 % o f l y m p h o c y te s CD56+ CD3+ (NKT cells) Bef ore -125 000 250 000 500 000 0 5 10 15 % o f ly m p h o c y te s

Figure 15. Flow cytometry analysis of migrated PBMC.

The graphs show the percentage of A) CD3+(T cells), B) CD4+ (Th cells), C) CD14+ (Monocytes), D) CD56+CD3- (NK cells), E) CD4dimCD25bright (Treg) and F) CD56+CD3+ (NKT cells) before and after migration in each set up (n=3) (note the different scale on y-axis). The graphs show the mean and SD.

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21 A B C D E F CD3+ (T cells) 125 000 MØ 250 000 MØ 500 000 MØ -30 -20 -10 0 10 p e rc e n til e fo ld c h a n g e CD4+ (Th cells) 125 000 MØ 250 000 MØ 500 000 MØ -10 0 10 20 p e rc e n til e fo ld c h a n g e CD14+ (Monocytes) 125 000 MØ 250 000 MØ 500 000 MØ -10 0 10 20 30 40 50 p< 0.05 p< 0.01 p< 0.01 p e rc e n til e fo ld c h a n g e CD56+ CD3- (NK cells) 125 000 MØ 250 000 MØ 500 000 MØ -30 -20 -10 0 10 p e rc e n til e fo ld c h a n g e CD4+ CD25+ (Treg) 125 000 MØ 250 000 MØ 500 000 MØ -5.0 -2.5 0.0 2.5 5.0 p e rc e n til e fo ld c h a n g e CD56+ CD3+ (NKT cells) 125 000 MØ 250 000 MØ 500 000 MØ -30 -20 -10 0 10 p e rc e n til e fo ld c h a n g e

Figure 16. Percentile fold change of migrated PBMC into wells containing 1.25x105,

2.5x105or 5x105 alternatively activated macrophages compared to wells containing medium

alone. A) CD3+(T cells), B) CD4+ (Th cells), C) CD14+ (Monocytes), D) CD56+CD3- (NK cells), E) CD4dimCD25bright (Treg) and F) CD56+CD3+ (NKT cells) (n=3) (note the different scales on y-axis). The results showed a significant (p<0.01) increase in monocytes that migrated into wells containing alternatively activated macrophages compared to medium alone. The graph shows mean and SD.

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5.2.3 Trophoblast cell line

These experiments investigated the recruiting ability of the trophoblast cell line HTR-8. 5x105 PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:5 and the lower wells contained either medium alone or medium with 7.5x104, 1.25x105 or 2.5x105 trophoblasts (Figure 17). CXCL12 was used as a positive control.

No significant difference was observed when comparing the amount of PBMC recruited by trophoblasts as compared to medium alone, neither was there a significant difference when comparing the positive control wells (CXCL12) with medium alone.

The composition of PBMC was investigated using flow cytometry (Figure 18 and 19). The results showed that significantly (p<0.01) more monocytes migrated into wells containing trophoblasts compared to medium alone. Further it was shown that significantly fewer Th cells migrated into wells containing trophoblasts compared to medium alone.

CX CL12 -75.000 125.000 250.000 0 25000 50000 75000 100000 No . o f m ig ra te d P B M C

Figure 17. 5x105 PBMC migrated ON through Matrigel-coated transwell inserts. The Matrigel concentration used was 1:5 and the lower wells contained either medium alone or medium with 7.5x104, 1.25x105 or 2.5x105 trophoblasts (n=4). No significant difference was observed when comparing the amount of PBMC recruited by trophoblasts as compared with medium alone. Each well concentration was run in triplicates. The graph shows mean and SEM.

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23 A B C D E F G H CD3+ (T cells) Bef ore -75 0 00 125 000 250 000 0 10 20 30 40 50 60 70 80 % o f l ym p h o cy te s CD4+ (Th cells) Bef ore -75 00 0 125 000 250 000 0 10 20 30 40 50 60 70 80 % o f l ym p h o cy te s CD14+ (Monocytes) Bef ore -75 00 0 125 000 250 000 0 10 20 30 40 50 60 70 80 % o f P B M C CD56+CD3- (NK cells) Bef ore -75 00 0 125 000 250 000 0 10 20 30 40 50 60 70 80 % o f l ym p h o cy te s CD4+CD25+ (Treg) Befo re -75 0 00 125 0 00 250 0 00 0 1 2 3 4 5 % o f l ym p h o cy te s CD56+CD3+ (NKT cells) Bef ore -75 0 00 125 000 250 000 0 1 2 3 4 5 % o f l ym p h o cy te s CD8+ (Tc cells) Bef ore -75 00 0 125 000 250 000 0 10 20 30 40 50 60 70 80 % o f l ym p h o cy te s CD19+ (B cells) Befo re -75 000 125 000 250 000 0 5 10 15 20 % o f l ym p h o cy te s

Figure 18. Flow cytometry analysis of migrated PBMC.

The graphs show the percentage of A) CD3+(T cells), B) CD4+ (Th cells), C) CD14+ (Monocytes), D), CD56+CD3- (NK cells) E) CD4dimCD25bright (Treg) and F) CD56+CD3+ (NKT cells), G) CD8+ (Tc cells), H) CD19+ (B cells) before and after migration in each set up (n=3) (note the different scales on y-axis). The graphs show the mean and SD.

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24 A B C D E F G H CD3+ (T cells) 75 000 125 000 250 000 -10 0 10 20 30 p e rc e n til e fo ld c h a n g e CD4+ (Th cells) 75 000 125 000 250 000 -10 0 10 20 30 p e rc e n til e fo ld c h a n g e CD14+ (M onocyte s) 75 000 125 000 250 000 -10 0 10 20 30 40 50 p<0.01 p<0.01 p<0.01 p e rc e n til e fo ld c h a n g e CD56+CD3- (NK cells) 75 000 125 000 250 000 -30 -20 -10 0 10 p e rc e n til e fo ld c h a n g e CD4+CD25+ (Treg) 75 000 125 000 250 000 -5.0 -2.5 0.0 2.5 5.0 p e rc e n til e fo ld c h a n g e CD56+CD3+ (NKT cells) 75 000 125 000 250 000 -5.0 -2.5 0.0 2.5 5.0 p e rc e n til e fo ld c h a n g e CD8+ (Tc cells) 75 000 125 000 250 000 -30 -20 -10 0 10 P<0.05 P<0.01 P<0.01 p e rc e n til e fo ld c h a n g e CD19+ (B cells) 75 000 125 000 250 000 -5 0 5 10 p e rc e n til e fo ld c h a n g e

Figure 19. Percentile fold change of migrated PBMC into wells containing 7.5x104, 1.25x105 or 2.5x105 trophoblasts. A) CD3+( T cells), B) CD4+ (Th cells), C) CD14+ (Monocytes), D) CD56+CD3- (NK cells), E) CD4dimCD25bright (Treg) and F) CD56+CD3+ (NKT cells) G) CD8+ (Tc cells), H) CD19+ (B cells) (n=3) (note the different scales on y-axis). The results showed a significant (p<0.01) increase in monocytes that migrated into wells containing trophoblasts. The graph shows mean and SD.

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6 Discussion

The results from this study show that macrophages, classically and alternatively activated, have a recruiting ability. Further, there is indication of trophoblasts also holding this ability. The primary cell population recruited by the macrophages and trophoblasts were monocytes. This type of research is important for the understanding of cell recruitment to the foetal-maternal interface during pregnancy, where a non-functional recruitment of important cell populations can result in pregnancy complications.

The first part of this study involved establishing a migration assay which would allow selective migration of PBMC in a transwell system. This type of set-up is common when evaluating recruitment ability of different cell types and chemokines. However, there are many parameters that can affect the migration like transwell pore size, Matrigel concentration, cell/chemokine concentrations and time. Although the design of the experiments was based on information reported in the literature, it should be noted that the conditions used vary a lot, and also, each parameter has to be tested in one owns laboratory environment. Therefore several different combinations were tried before a satisfactory result was seen.

The pore size 5 µm was chosen early on and was the only parameter that was not changed. This pore size along with 8 µm is commonly used in these kinds of experiments (Graham et al., 1998; Felkel et al., 2001).

Initially, cells were allowed to migrate through uncoated transwell inserts and chemokines, with well known recruiting ability for specific cell types (Table 1), were used as recruiting stimuli. No selective migration could be seen, neither when comparing the amount of cells recruited by chemokines nor when looking at which cell types the various chemokines had recruited. One of the chemokines, CCL2, has been shown to induce selective migration of monocytes at concentrations from about 4.5 ng/ml (Renaud et al., 2009) indicating that the absence of selective migration in this study should not be the result of a too low chemokine concentration, since the concentration used was 100 ng/ml.

Matrigel-coated transwell inserts where thereafter used. The idea was that a small pore size along with the Matrigel should make it harder for the cells to migrate and reduce the risk of spontaneous migration. Matrigel concentrations used for migration assays with the purpose of studying recruitment ability of cells or chemokines generally have a lower concentration than those that are used for studying invasion, i.e. ability to invade tissue or to visualize the invasion in 3D (Felkel et al., 2001; Tarrade et al., 2001). This is because a lower Matrigel concentration results in a protein coating of the transwell inserts whereas a higher Matrigel concentration results in a gel and therefore is both thicker and exerts more resistance on migrating cells. Since these experiments aimed to investigate the recruitment ability of macrophages and trophoblasts, a lower concentration of Matrigel was used. A lower concentration also allowed enough cells, for flow cytometry analysis, to migrate into the lower wells.

The Matrigel concentration 1:7.5 was initially used based on literature (Renaud et al., 2009) and on the fact that it, based on our initial Matrigel titration experiment (data not shown), allowed sufficient amounts of cells to migrate. Several different recruitment stimuli, cells and chemokines, were used to test if this Matrigel concentration along with other parameters would allow selective recruitment of cells. However, in most experiments no selective migration could be seen. In the experiments that gave an indication of selective migration the

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results were often weaker when repeating the experiment (Figure 4 and 7). In retrospect, the experiment using CXCL12 as recruiting stimuli (Figure 7) appeared to be promising and it is now tempting to speculate that if repeating the experiment a significant selective migration could have been seen. However, it should not be overlooked that when using alternatively activated macrophages (Figure 4) or in particular classically activated macrophages (Figure 6) the results were inconsistent.

The Matrigel concentration was thereafter changed to 1:5, with the expectation that a higher concentration would reduce the spontaneous migration. Initially no such change was seen (Figure 8), but later when other parameters like number of recruiting cells was changed a significant selective migration was seen (Figure 10). In contrast, a Matrigel concentration of 1:2 showed almost no migration of cells (Figure 9), confirming that such a high concentration forms a solid gel, which was not suitable in this context.

Migration time is another parameter that varies greatly in the literature, the time vary from 1 h (Felkel et al., 2001) to 24 h (Graham et al., 1998). In this study migration for 2 h and ON was tried, ON was later chosen since selective migration was seen and rare cell populations, e.g. Tregs, would be found in sufficient amount for flow cytometry analysis if they migrated towards the recruiting stimuli.

Further, the number of cells placed into the transwell inserts also varies greatly in the literature, from 2x104 (Kilburn et al., 2000) to 1x106 (Huang et al., 2008). In this study 1x106 PBMC were originally used but the cell number was later reduced to 5x105 PBMC during the method development. Since we did not use isolated cell populations but instead PBMC, a larger number of migrating cells was chosen. A larger number of PBMC also allowed flow cytometry analysis and the possibility to analyse rare cell populations which would not be possible if the initial PBMC number would have been very low. The reduction of cell number from 1x106 to 5x105 PBMC was made in order to reduce the spontaneous migration. It is hard to say to which extent the reduction of migrating cells played in that the final migration assay set-up showed selective migration, since this was not the only parameter changed.

To sum up this part of the project, selective migration could be shown when 5x105 PBMC were allowed to migrate ON through transwell inserts coated with Matrigel, concentration 1:5, into wells containing 250 ng/ml CXCL12 which was used as a positive control (Figure 10).

With a functional migration assay, the aim was to investigate the recruitment ability of dMØ and trophoblasts. The hypothesis was that dMØ and trophoblasts are involved in recruiting monocytes and NK cells to the decidua during pregnancy.

First the recruitment ability of classically and alternatively activated macrophages was investigated. Selective migration could be seen when comparing the recruitment ability of 1.25x105 classically activated macrophages to medium alone (Figure 11) and when comparing 5x106 alternatively activated macrophages to medium alone (Figure 14). A significant recruitment of monocytes by alternatively activated macrophages was seen (Figure 15 and 16) and there was an indication that classically activated macrophages also recruited monocytes (Figure 12 and 13), although this needs to be confirmed by repeating the experiment.

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Peripheral blood monocytes migrate into tissues upon stimuli; they are thereafter educated and activated by the local microenvironment (Nagamatzu and Schust, 2010). Classically activated macrophages are known to secrete pro-inflammatory chemokines and to be involved in cell-mediated immunity (promote inflammation), whereas alternatively activated macrophages are involved in tissue repair and humoral immunity (anti-inflammatory responses) (Gordon and Taylor, 2005). Classically activated macrophages have been shown to produce CCL2, which recruits monocytes and macrophages to sites of infection by binding the CCL2 receptor (Hori et al., 2008). Further, classically activated macrophages produce chemokines which recruit Th1, Tc and NK cells (Mantovani et al., 2004). No recruitment of Th or NK cells was seen in this study (Figure 12 and 13); and Tc cells were not analyzed. dMØ secrete IL-15 (Nagamatzu and Schust, 2010) a chemokine known to attract NK cells (Allavena et al., 1997). CCL2 is also expressed by these macrophages and is known to attract monocytes (Gustafsson et al., 2008), further dMØ have been shown to secrete CCL18 known to foremost attract naive T cells (Adema et al., 1997) and Th2 cells (de Nadai et al., 2006). The results from this study shows that alternatively activated macrophages can recruit PBMC (Figure 14) and it also showed selective migration of monocytes into wells containing these macrophages. The in vitro alternatively activated macrophages used in this study are phenotypically similar to dMØ, based on that they express many of the same surface markers (Svensson et al. – unpublished data), but since the chemokine profile of these in vitro macrophages has not been investigated so far, we do not know if they secrete the appropriate chemokines mentioned above and thus can attract the same cell populations as dMØ. The last cell type investigated for its recruiting ability in these experiments was trophoblasts, in this case represented by a cell line of human origin. The recruitment ability of trophoblasts is well documented. Tumour necrosis factor (TNF) –α, expressed by a variety of maternal and foetal tissues (Hunt et al., 1996), increases the trophoblasts’ secretion of CCL2 and thereby the trophoblasts’ ability to recruit monocytes to the foetal-maternal interface (Renaud et al., 2009). Trophoblasts also secrete the chemokine CXCL16, which has been shown to attract T cells and monocytes through the CXCR6 receptor expressed by these cells (Huang et al., 2008). Further macrophage inflammatory protein (MIP) -1α is also secreted by primary trophoblasts and has been shown to recruit both dNK cells and monocytes to the foetal-maternal interface (Drake et al., 2001).

The results from this study showed selective recruitment of monocytes by trophoblasts (Figure 18C and 19C), but selective migration was not seen for any other cell type investigated. In vivo, macrophages are found in the decidua basalis in the vicinity of trophoblast populations. Macrophages are known to have receptors for many of the ligands found on trophoblasts, but the exact result of their interaction is not well understood (Renaud and Graham, 2008).

The most common cell population in the decidua during early pregnancy is dNK cells; these cells constitute ~70 % of the leukocytes (Renaud and Graham, 2008). The chemokine CXCL12 is constitutively produced by trophoblasts and has been shown to recruit dNK cells through the CXCL12 specific receptor CXCR4 expressed by dNK cells (Wu et al., 2005). In this study no specific recruitment of NK cells by trophoblasts could be seen (Figure 18 and 19). A possible explanation is that the trophoblasts did not produce sufficient levels of CXCL12 during the duration of this experiment. One would have expected to see an increased migration of NK cell into wells containing CXCL12, but this was not seen (data not shown).

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CXCL12 secretion by trophoblasts has been shown in primary trophoblasts (Hanna et al., 2003; Wu et al., 2005), but to my knowledge CXCL12 secretion by the trophoblasts cell line HTR-8 has not been investigated. Another issue worth considering is the high spontaneous migration of NK cells observed in all experiments, which might have masked the potential specific recruitment of NK cells by macrophages and trophoblasts.

Finally, no specific recruitment of Treg cells, by alternatively activated macrophages or trophoblasts, could be seen. Tregs have been proposed to play an important role during pregnancy (Sasaki et al., 2004). They are known suppressors of the adaptive immune system and involved in self-tolerance. Further, Tregs have been shown to mediate alternative activation of macrophages (Tiemessen et al., 2007).

In vitro experiments are in general very simplified. For example, few studies have evaluated

the ability of a recruitment stimulus on PBMC, but instead isolated cell types have been used. Here we chose to use PBMC since this is a more biological approach, more similar to the in

vivo situation. At this point only one cell type has been used as recruiting stimulus, but it

would be interesting to study the effect of combinations of cells, e.g. macrophages and trophoblasts. Further, it would be interesting to study the recruiting ability of NK cells. Still, it is hard to evaluate the in vivo function when only looking at the interaction between two or a few cell types, and it is thus hard to predict the actual function of different cell populations and chemokines, and get a correct picture of the complex interaction involving different cell types and chemokines at the foetal-maternal interface.

Further experiments involve to first test the recruiting ability of dMØ. It would also be interesting to collect the supernatants from decidual and trophoblast cells, combined or separately. The chemoattractive properties of the supernatants could then be investigated in the transwell system. This approach would present a more in vivo-like situation and would show if the decidual or trophoblast cells, combined or separately, are needed to recruit specific cell populations. Further, candidate molecules can be blocked and the resulting effect can be assessed. This type of research is important to understand the biology of normal pregnancy, but also to investigate crucial molecules which can be involved in the development of pregnancy complications. For example, a defective recruitment of macrophages could hazard the immunosuppressive environment and a defective recruitment of NK cells could impair the formation of spiral arteries.

In conclusion, this study presents, after testing several conditions, a protocol that seems useful for assessing the chemotactic properties of cells relevant for studies in reproductive immunology. Although the experiments need to be repeated and extended before certain conclusions can be drawn, preliminary results showed that macrophages and trophoblasts are primarily involved in the recruitment of monocytes, whereas recruitment of NK cells appears to involve other mechanisms.

7 Acknowledgements

A warm thanks to my laboratory supervisor Judit Svensson, for all the support that I have received and for answering a thousand questions. Jan Ernerudh, my supervisor, for enthusiasm and help and Sofia Freland for all constructive criticising and pushing me forward with writing this thesis. Finally, big thanks to all personnel at the unit for Autoimmunity and immune Regulation for a great working climate and for donating blood.

References

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