Quantification of uterine and placental cytokines and chemokines in syngeneic pregnancy (day 16.5 <i>pc</i>).

<p>Uterine and placental tissues were prepared as described in Materials and Methods : (A) Uteri from NP mice, (B) Uteri from <b>syngeneic</b> pregnancy (day 16.5 d<i>pc</i>), (C) Placenta from <b>syngeneic</b> pregnancy (same animals). Samples were analyzed simultaneously for the following 22 cytokines: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, CSF2 (GM-CSF), CSF3 (G-CSF), IFNγ, CXCL10 (IP-10), CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), TNF-α. Only IL-9, IL-10, GM-CSF (CSF2), G-CSF (CSF3), CXCL10 (IP-10), IL-1α, CCL3 (MIP-1α), CXCL1 (KC), CCL2 (MCP-1) yielded reproducibly significant measurements and have been presented. Striped bars: whole organ, black bars: enriched leukocytes from the same organ. Data are from 4 to 5 different samples. Statistically significant differences: (*) p≤0.05, (***) p≤0.001.</p

Antibody and dilutions used for flow cytometry staining.

<p>NA: not applicable.</p><p>Antibody and dilutions used for flow cytometry staining.</p

Analyses of immune cell populations present in the uterus of NP or pregnant mice, and in placenta (16.5 d<i>pc</i>).

<p>Only viable cells excluding propidium iodide were analysed. Enriched leukocytes from NP (A,B) or pregnant (16.5 d<i>pc</i>) uterus (C,D) and placenta (E,F) were analysed by flow cytometry. R2-gated cells (left, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g002" target="_blank">Figure 2</a>) were analysed on the basis of the following cell surface markers: TCRβ⁺/CD4⁺ (CD4 T cells); TCRβ⁺/CD8⁺ (CD8 T cells); NK1.1⁺/TCRβ⁻ (NK cells); TCRβ⁺/NK1.1⁺ (NKT cells); CD19⁺/B220⁺ (B cells); Gr1⁺/CD11b⁺ (myeloid Gr1+ cells including monocytes); Gr1^−/CD11c⁺/CD11b^Hi/low (myeloid CD11c+ cells including DCs). R1-gated cells (right, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g002" target="_blank">Figure 2</a>) were analysed on the basis of the following cell surface markers: Gr1⁺/CD11b⁺ (Granulocytes); CD11b^Hi/Gr1⁺/Gr1^+/−/CD11c^+/− (Highly granulosity cells or HGC). The results are representative from a typical experiment of a pool of 7 mice (A, B) or from a single mouse (C, D, E, F). The experiment was repeated at least twice with 3–6 animals per assay.</p

High granulosity cells in non pregnant uterus are primarily eosinophils.

<p>Viable R1- or R2- gated CD45.2+ cells from NP uterus were analysed by FACS stained for APC and granulocytes markers (CD11b, CD24, CD11b, F4/80, Ly6C, Ly6G, MHC class II and CCR2) (A, B and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-t001" target="_blank">Table 1</a>) and for NK and T cell markers (CD11b, NK1.1, CD3ε, CD8α and CD4) (B and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-t001" target="_blank">Table 1</a>). The experiment was repeated twice with 6 females each time (A, B). Viable CD45.2+, CD11b+, F4/80+ R1 (C) or R2 (D) cells were sorted by flow cytometry and spun onto Super + glass microscope slides using a Cytospin cytofuge. Cells were fixed with methanol and stained with Wright-Giemsa. Pictures were taken on a Nikon H600L microscope equipped with a DS-Fi2-Nikon camera (C, D). The experiment was performed twice with at least 5 females each time.</p

Purity of leukocytes and presence of fetal cells after enrichment from placenta and uterus (16.5 d<i>pc</i>).

<p>A. Enriched leukocyte preparations from non-pregnant (NP) uterus, pregnant (16.5 d<i>pc</i>) uterus (P) and placenta were analyzed by flow cytometry. Viable cells excluding propidium iodide were gated on the basis of forward (FSC) and side scatter (SSC) criteria. The average leukocyte purity assessed by the presence of the CD45.2 marker was 89.3% for NP uterus, 97% for pregnant uterus, and 95% for placenta, compared to 98% for the spleen (positive control for the assessment of purity) from the same mice. Analyses were performed on at least 3 animals per group B. CD45.2+ B6 females were crossed with CD45.1+ B6 males. CD45+ leukocyte populations from the uterus and placenta were analyzed by flow cytometry on day 16.5 <i>pc</i>. The detection of CD45.1+2+ cells revealed the presence of a small percentage (<5%) of fetal leukocytes. The experiment has been repeated 3 times.</p

Cytokine and chemokine expression levels in uterus from NP mice versus syngeneic or allogeneic pregnancies (day 16.5 <i>pc</i>).

<p>The same protocol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g007" target="_blank">Figure 7</a> was followed. Proteins were measured from whole uterus (A), or from enriched uterine leukocytes (B) from NP uterus, or syngeneic or allogeneic pregnancies (note the important scale variations). Striped bars: NP uterus, grey bars: syngeneic pregnancy at 16.5 d<i>pc</i>, black bars: allogeneic pregnancy at 16.5 d<i>pc</i>. Data are from 4 to 5 different samples. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.</p

Cytokine and chemokine expression levels in placentae from syngeneic or allogeneic pregnancies (day 16.5 <i>pc</i>).

<p>Analyses were performed on the placentas from the same pregnant mice as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g008" target="_blank">Figure 8</a>. Proteins were measured from whole placenta (A), or from enriched placental leukocytes (B) from syngeneic or allogeneic pregnancies (note the important scale variations). Grey bars: syngeneic pregnancy, black bars: allogeneic pregnancy. Data are from 4 to 5 different samples. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.</p

Drastic changes in the scatter distribution of leukocytes from NP, or pregnant uterus, and placenta at various stages of a syngeneic pregnancy.

<p>The same protocol as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107267#pone-0107267-g001" target="_blank">Figure 1</a> was followed. Viable cells excluding propidium iodide were gated on the basis of forward (FSC) and side scatter (SSC) criteria, from uterus (A) or placenta (D). Mean percentages (B, E) and total cell numbers (C, F) in « granular » (R1 gate, white bars) or « lymphoïd/monocytoïd » (R2 gate, black bars) are presented. NP uteri were pooled from 4 to 15 mice in all phases of œstrus cycle and the experiment was repeated 6 times. At each stage of pregnancy or post-partum, the data were collected from 4 to 14 mice assayed in 2 to 9 separate experiments. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.</p

AKT Phosphorylation in Cells during Apoptosis of C. trachomatis-Infected Cells

<p>Cells were infected with C. trachomatis at an MOI of 1.0 for 24 or 48 h, or mock-infected, and then incubated with 50 μM LY294002 (LY) or control buffer for 6 h before treating with control buffer or 2 μM STS overnight. Cells were then collected for Western immunoblotting and analyzed for phosphorylation of AKT on residue Ser⁴⁷³, as described in Materials and Methods. STS treatment by itself did not affect AKT Ser⁴⁷³ phosphorylation in infected cells, but LY294002 caused AKT Ser⁴⁷³ to become partially dephosphorylated. This residue became dephosphorylated almost completely when LY294002 was combined with STS in infected cells. One experiment of two representative experiments performed on separate days is shown.</p

Effect of PI3K on BAD Phosphorylation during C. trachomatis Infection

<p>Cells over-expressing BAD were infected with C. trachomatis at an MOI of 1.0 for 26 h or mock-infected, and then incubated with 50 μM LY294002 (LY) or control buffer for 6 h before treating with 2 μM STS overnight. Cells were then collected for Western immunoblotting and analyzed for total BAD protein (top row), phosphorylation of BAD on residue Ser¹³⁶ (middle row), or total P42 MAP kinase protein (bottom row), as described in Materials and Methods. C. trachomatis infection led to a decrease in the level of BAD, which was further decreased by STS treatment. Pre-incubation with LY294002 did not further alter the level of total BAD (top row). STS-treatment of uninfected cells caused complete dephosphorylation of BAD, but BAD remained phosphorylated after STS-treatment of infected cells (middle row). However, pre-incubation of infected cells with LY294002 before treatment with STS resulted in complete dephosphorylation of BAD (middle row). Infection had no effect on total P42 MAP kinase protein levels (bottom row). One experiment of three representative experiments performed on separate days is shown.</p