Differential expression of collectins in human placenta and role in inflammation during spontaneous Labor.

© 2014 Yadav et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Collectins, collagen-containing Ca2+ dependent C-type lectins and a class of secretory proteins including SP-A, SP-D and MBL, are integral to immunomodulation and innate immune defense. In the present study, we aimed to investigate their placental transcript synthesis, labor associated differential expression and localization at feto-maternal interface, and their functional implication in spontaneous labor. The study involved using feto-maternal interface (placental/decidual tissues) from two groups of healthy pregnant women at term (≥37 weeks of gestation), undergoing either elective C-section with no labor ('NLc' group, n = 5), or normal vaginal delivery with spontaneous labor ('SLv' group, n = 5). The immune function of SP-D, on term placental explants, was analyzed for cytokine profile using multiplexed cytokine array. SP-A, SP-D and MBL transcripts were observed in the term placenta. The 'SLv' group showed significant up-regulation of SP-D (p = 0.001), and down-regulation of SP-A (p = 0.005), transcripts and protein compared to the 'NLc' group. Significant increase in 43 kDa and 50 kDa SP-D forms in placental and decidual tissues was associated with the spontaneous labor (p<0.05). In addition, the MMP-9-cleaved form of SP-D (25 kDa) was significantly higher in the placentae of 'SLv' group compared to the 'NLc' group (p = 0.002). Labor associated cytokines IL-1α, IL-1β, IL-6, IL-8, IL-10, TNF-α and MCP-1 showed significant increase (p<0.05) in a dose dependent manner in the placental explants treated with nSP-D and rhSP-D. In conclusion, the study emphasizes that SP-A and SP-D proteins associate with the spontaneous labor and SP-D plausibly contributes to the pro-inflammatory immune milieu of feto-maternal tissues.Funding provided by BT/PR15227/BRB/10/906/2011) Department of Biotechnology (DBT), Government of India http://dbtindia.nic.in/index.asp (TM) and Indian Council of Medical Research (ICMR) Junior Research Fellowship (JRF)/Senior Research Fellowship (SRF), Government of India, www.icmr.nic.in (AKY)

Immunolocalization of SP-A and MBL proteins at feto-maternal interface (n = 5/group).

<p>The 5 µm thin tissue sections a) placenta or c) decidua were probed with monoclonal antibody to SP-A. Anti-MBL monoclonal antibody was applied to b) placenta or d) decidua. e) Negative controls for the respective tissues. The tissue sections were counterstained with hematoxylin and mounted with DPX. Representative images are 400× original magnification. f) The relative intensity of SP-A and MBL staining was quantitated by NIS elements version 4.1 software analysis tools. Bars are mean ±SEM, *p<0.05 and **p>0.05.</p

Details of primer pair sequences, annealing temperature and product length for amplification of each transcript.

<p>Details of primer pair sequences, annealing temperature and product length for amplification of each transcript.</p

Differential expression of SP-A, SP-D and MBL in term placenta

<p>(n = 5/group). A) Semi-quantitation of the transcripts assessed by real time RT-PCR between two groups of placenta from caesarean section with no labor (‘NLc’) and placenta from normal vaginal delivery with spontaneous labor (‘SLv’). The experiments were carried out 3 times for each transcript and relative quantity was calculated by 2^−ΔΔCt method after normalizing with 18S rRNA of the respective sample. The relative fold expression presented is compared to controls; error bars represent ±SEM for each transcript. B) Western blot analysis of SP-A, SP-D and MBL in total placental protein (30 µg) of both groups (‘NLc’ vs ‘SLv’; n = 5/group). The blots were probed with mouse monoclonal antibody to SP-A (i), SP-D (ii), MBL (iii), and rabbit polyclonal antibody to β-actin for normalization (iv). C) Densitometric analysis of blots for SP-A, SP-D and MBL proteins using Labscan and ImageQuant TL analysis tools (GE Healthcare, USA) and presented as mean ±SEM; i) SP-A, ii) SP-D and iii) MBL. Statistical analysis was performed with one way ANOVA at p<0.05.</p

Immunolocalization of SP-D at the term feto-maternal interface (n = 5/group).

<p>The 5 µm thin tissue sections were probed with monoclonal antibody to SP-D a) placenta (‘NLc’ vs. ‘SLv’); b) decidua (‘DNLc’ vs. ‘DSLv’) and subsequently counter stained with hematoxylin. Representative images are 400× original magnification (a) or 200× original magnification (b), insets are negative controls for the respective tissues. c) The relative intensity of SP-D staining in two groups of placenta and decidua was quantitated by NIS elements version 4.1 software analysis tools. Bars are mean ±SEM; *p<0.05.</p

Western blot analysis of SP-D in term decidual tissues (‘DNLc’ vs. ‘DSLv’).

<p>Total term decidual proteins (30 µg each sample) were blotted and probed with mouse monoclonal antibody to SP-D or polyclonal antibody to β-actin; A) representative images (n = 5/group) B) Relative quantity of SP-D in each group was obtained by Labscan and 1D ImageQuant analysis tools using β-actin as internal reference control for normalization. Bars are mean ±SEM; *p<0.05.</p

Schematic diagram of plausible functions of SP-A and SP-D at the term feto-maternal interface during labor.

<p>Schematic diagram of plausible functions of SP-A and SP-D at the term feto-maternal interface during labor.</p