Relative quantification comparisons of CX43 and OXTR in ULTR cells treated with 5, 10 and 20 μg/ml of rhSP-A (A-B) and rhSP-D (C-D) after 4 and 6 h (*p<0.05, **p<0.01, ***p<0.001).

<p>rhSP-A led to an increase of CX43 mRNA expression after 4 h at a concentration of 10 and 20 μg/ml, effect that seemed to disappear after 6 h, and of OXTR after 6 h at concentrations of 10 and 20 μg/ml. rhSP-D resulted in an increase of CX43 transcript at all concentrations, and of OXTR transcript after 6 h at a concentration of 5 μg/ml (n = 3). Letter denotes values significantly different from each other as indicated by the horizontal and vertical lines (p<0.05).</p

Primer sequences of human gene targets used for qPCR experiments.

<p>Primer sequences of human gene targets used for qPCR experiments.</p

Relative quantification comparisons of SP-A1 (A), SP-A2 (B), and SP-D (C) in ULTR treated with 5, 10 and 20 μg/ml of rhSPD after 6 and 12h (*p<0.05, **p<0.01, ***p<0.001).

<p>rhSP-D treatments resulted in an increase of SP-A1, SP-A2 and SP-D mRNA expression at 6 h at 5 and 10 μg/ml. The effect was inverted at 12 h, with rhSP-D leading to a decrease in the expression of the above genes (n = 3).</p

Multiplex cytokine array analysis of the supernatants of ULTR cells treated with rhSPA or rhSPD at different concentration time points (n = 2).

<p>Secretion levels for GROα (A), IL-6 (B), IL-6 Ra (C) and IL-8 (D), were measured by a MagPix Milliplex kit (EMD Millipore) rhSP-A treatments (10 and 20 μg/ml) caused an increase in the expression of GROα and IL-8 compared to the untreated cells, but did not have a profound effect on IL-6 and IL-6Ra. rhSP-D treated cells (10 and 20 μg/ml) appeared to express higher levels of GROα, IL-6 and IL-8 at 12 and 24 h compared to the untreated cells. ULTR cells treated with rhSP-D (10 and 20 μg/ml) expressed higher levels of IL-6 Ra after 24h. (*p<0.05, **p<0.01, ***p<0.001). Letter denotes values significantly different from each other as indicated by the horizontal and vertical lines (p<0.05).</p

Immunofluorescent analysis of ULTRs, immunostained for SP-A (A) and SP-D (D).

<p>Nuclear staining using phalloidin 488 for SP-A (B) and SP-D (E). Merged images for SP-A (C) and SP-D (F). x 40 magnification. (G) and (H): Representative cells immunostained for SP-A and SP-D respectively. Merged image (I) showing the comparison of the fluorescence intensity of the cells between SP-A (red) and SP-D (black). SP-D immunostained cells appeared to have higher intensity compared to SP-A immunostained cells.</p

Surface disc area comparisons between myometrium cells growing in collagen with or without treatment with rhSP-A or rhSP-D, after 3 and 24 h.

<p>Cells were seeded at a specific density and were grown in a collagen matrix to mimic a 3D milieu. rhSP-A and rhSP-D significantly induced a similar contractility of ULTR cells compared to untreated cells, at 3 and 24 h (A), (*p<0.05, **p<0.01, ***p<0.001). There was no significance in between treatments or time points. Representative collagen discs of 3 h treatment (B; NS: no supplement). Insert: image of ULTRs grown within the collagen matrix (n = 4).</p

Surfactant Proteins SP-A and SP-D Modulate Uterine Contractile Events in ULTR Myometrial Cell Line

<div><p>Pulmonary surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules. However, there is extrapulmonary existence, especially in the amniotic fluid and at the feto-maternal interface. There is sufficient evidence to suggest that SP-A and SP-D are involved in the initiation of labour. This is of great importance given that preterm birth is associated with increased mortality and morbidity. In this study, we investigated the effects of recombinant forms of SP-A and SP-D (rhSP-A and rhSP-D, the comprising of trimeric lectin domain) on contractile events <i>in vitro</i>, using a human myometrial cell line (ULTR) as an experimental model. Treatment with rhSP-A or rhSP-D increased the cell velocity, distance travelled and displacement by ULTR cells. rhSP-A and rhSP-D also affected the contractile response of ULTRs when grown on collagen matrices showing reduced surface area. We investigated this effect further by measuring contractility-associated protein (CAP) genes. Treatment with rhSP-A and rhSP-D induced expression of oxytocin receptor (OXTR) and connexin 43 (CX43). In addition, rhSP-A and rhSP-D were able to induce secretion of GROα and IL-8. rhSP-D also induced the expression of IL-6 and IL-6 Ra. We provide evidence that SP-A and SP-D play a key role in modulating events prior to labour by reconditioning the human myometrium and in inducing CAP genes and pro-inflammatory cytokines thus shifting the uterus from a quiescent state to a contractile one.</p></div

Representation of the average distance (A), velocity (B) and displacement (C) comparisons in ULTR treated with and without rhSP-A and rhSP-D (*p<0.05, **p<0.01, ***p<0.001).

<p>Cell movements (n = 25 cells, from a single experiment) were observed either without treatment or with treatment with surfactant proteins (10 μg/ml). Both treatments led to a significant increase in velocity, distance travelled and their displacement from their initial position compared to untreated cells. rhSP-A treatment led to a significant increase in velocity and distance compared to rhSP-D treated cells. There was no apparent significance in displacement in between treatments.</p