Shanmuga Priyaa Madhukaran Jawaharlal Nehru Institute for Advanced Studies (JNIAS), Secunderabad, India
[email protected]
Taruna Madan Gupta National Institute for Research in Reproductive Health, Mumbai, India
[email protected]
Uday Kishore Brunel University, United Kingdom
[email protected]
SURFACTANT PROTEIN A AND D ALTER LPS MEDIATED PRO-‐INFLAMMATORY EFFECTS ON DECIDUAL MACROPHAGES AND COULD BE RELEVANT IN PARTURITION
Antigen Retrieval
Observed under the microscope
Decidua
Paraffin embedded
Immunohistochemistry Flow cytometry
Anti-‐mouse TNF-‐α FITC
Confocal DAPI Analyzed by Microscopy two-‐color FACS
Results SP-‐A & SP-‐D mRNA in the murine decidua 0.06 17.5 dpc 19.5 dpc
a.
1 2 3 4 5 6 7 8 9 1.
SP-‐D (156 bp)
17.5 dpc
1 2 3 4 5 6 7 8 9 SP-‐A (225 bp)
Fig. 3. Immunohistochemical localization of SP-‐ A & SP-‐D in the decidua at 17.5 dpc (b) & (e) and 19.5 dpc (c) & (f). SP-‐A and SP-‐D (brown) were observed in the decidua stroma, and in and around the spiral artery and blood vessels. Intense SP-‐D (b) & (c) staining was detected at 17.5 and 19.5 dpc when compared to SP-‐A. Red arrow head indicate trophoblast cells. Hematoxylin counterstained cells appear blue.
SP-‐D (156 bp)
19.5 dpc
Fig. 2. Real time RT-‐PCR amplified products separated by agarose gel electrophoresis. Gene expression of SP-‐A & SP-‐D in the decidua at (1.)17.5 dpc and (2.)19.5 dpc. One μg total RNA in decidua was used for RT-‐PCR. Lane 1: Ladder (100bp); Lane 2: 18s; Lane 3: 18s NTC ; Lane 4: SP-‐A positive control (lung); Lane 5: SP-‐ A decidua; Lane 6: SP-‐A NTC; Lane 7: SP-‐D positive control (lung); Lane 8: SP-‐D decidua; Lane 9: SP-‐D NTC.
b.
c.
Fig.4.Confocal images on a Zeiss 510 meta microscope performed by counterstaining the decidual cells with DAPI. a) F4/80 positive cells (red); b) TNF-‐α expression (green) c) DAPI staining (blue). Effect of rhSP-‐A & rhSP-‐D in murine decidua at 17.5 dpc 30 F4/80 positive cells
25
TNF-‐alpha
20
10 5 0
Conclusion Decidual SP-‐A and SP-‐D may be playing a role in parturition by immunomodulation of DMs and are likely to be protective against intrauterine infection/ inflammation during pregnancy.
Acknowledgement We acknowledge the facilities provided by National Institute for Research in Reproductive Health, Indian Council of Medical Research and Department of Biotechnology, India for international travel support.
References
15
Collectins
LPS (100ng)+SP-‐D (10µg)
SP-‐A SP-‐D Collectins
SP-‐A (225 bp)
19.5 dpc SP-‐A (20x)
Intracellular cytokine assay by confocal microscopy
Fig. 1. Expression profile of SP-‐A & SP-‐D mRNA levels in the decidua at 17.5 & 19.5 dpc determined by real time RT-‐PCR. SP-‐A & SP-‐D mRNA levels were normalized to the level of 18s rRNA present in each sample. SP-‐A mRNA was not detectable in the decidua at 17.5 & 19.5 dpc. The levels of decidual SP-‐D mRNA at 17.5 dpc differ significantly from SP-‐D mRNA levels at 19.5 dpc. The data represent the standard error of the mean, n=3 animals. 18s (175 bp)
17.5 dpc SP-‐A (20x)
LPS (100ng)+SP-‐D (5µg)
0
Control SP-‐A (20x)
Blood vessel
SP-‐D (10µg) +LPS (100ng)
Placenta
Real time PCR
Mounted
0.02
Trophoblast infiltration
SP-‐D (5µg) +LPS (100ng)
Isolation of decidua
Permeabilised 0.1% saponin
f.
Maternal spiral artery
Fixed 4% PFA
Dehydrated
0.04
e.
SP-‐D (10µg)
Quantitative RT-‐PCR
2.
Pregnant female mice
Formalin fixed
Counter stained
18s (175 bp)
C57BL/6 male mice
Anti-‐F4/80 PE
d.
19. 5 dpc SP-‐D (20x)
SP-‐D (5µg)
CDNA synthesis Invitrogen Kit
17.5 dpc SP-‐D (20x)
Control SP-‐D (20x)
LPS (100ng)+SP-‐A (10µg)
x
Secondary antibody
Trophoblast infiltration
Maternal decidua
LPS (100ng)+SP-‐A (5µg)
SYBR Green
SP-‐A (5 or 10 μg) or SP-‐D (5 or 10 μg) or LPS (1 μg)
c.
Fetal maternal interface
Stimulated,1 hr
Primary antibody SP-‐A at 1:10000 SP-‐D at 1:2000
RNA
a.
Fetal side-‐placenta
SP-‐A (10µg) +LPS (100ng)
C57BL/6 female mice
Blocked
b.
SP-‐A (5µg) +LPS (100ng)
Isopropanol
Fig. 5. Effects of SP-‐A & SP-‐D on the production of TNF-‐α, pro-‐inflammatory cytokine at 17.5 dpc by intracellular cytokine assay. At 17.5 dpc decidual cell were isolated, purified and then stimulated with rhSP-‐A or rhSP-‐D (5 and 10μg) or LPS (100ng, derived E. coli strain 011:B4) for 1 hour. Percentage of F4/80 positive cells and TNF-‐α cytokine was measured by two color flow cytometry. LPS mediated down regulation of F4/80 positive cells was seen at 17.5 dpc (9%) while TNF-‐α was highly up regulated (2 fold) in the presence of LPS. Low dose of rhSP-‐ A (5μg) &high dose of rhSP-‐D (10μg) more significantly down regulates (more than 3 fold) TNF-‐α production by macrophages. Similar down regulatory effect was also seen when decidual cells were pretreated with LPS followed by low-‐dose rhSP-‐A /rhSP-‐D ((5μg).
SP-‐A (10µg)
Methods
Cells counted Haemocytometer
Localization of SP-‐A & SP-‐D in the decidua
SP-‐A (5µg)
ü Determine levels of SP-‐A and SP-‐D transcripts in C57BL/6 murine decidua at 17.5 and 19.5 dpc. ü Localize SP-‐A and SP-‐D in C57BL/6 murine decidua at 17.5 and 19.5 dpc. ü Evaluate the in vitro immunological effects of SP-‐A and SP-‐D on decidual macrophages.
Enzyme digestion
Dewaxed
Chloroform
Decidua
Objectives
Tissue
Results
LPS (100ng)
Homogenized In Trizol
Aim of the study To investigate the expression and immunomodulatory effects of SP-‐A and SP-‐D on decidual macrophages in the murine decidua at 17.5 and 19.5 dpc .
Tissue
Results
Control
Tissue
Relative amount of SP-‐A & SP-‐D mRNA
•Decidua that connects the maternal tissue and blood with fetal membrane is thought to play a central role in parturition via ‘’decidual activation’’1. • There are three major leukocyte populations present in the decidua: natural killer cells, macrophages and T cells2,3. • Surfactant protein A and D, members of the collectin family were initially described as major components of lung surfactant4. • Recently SP-‐A and SP-‐D expression was shown in several extra-‐pulmonary tissues, including salivary gland, pancreas, kidney, intestine, reproductive tissues5. • Amniotic fluid SP-‐A is implicated in parturition (mice and humans)6. • Neither the expression nor localization of SP-‐ A and SP-‐D has been examined in murine decidua at near-‐term (17.5) and term (19.5 dpc).
Methods
Percentage of F4/80 positive cells & TNF-‐α
Introduction
1. Casey ML, MacDonald PC 1988 Biomolecular processes in the initiation of parturition: decidual activation. Clin Obstet Gynecol 31:533-‐552. 2. Bulmer JN Lash GE 2005 Human uterine natural killer cells: reappraisal. Mol. Immunol. 42, 511–521. 3. Moffett-‐King A 2002 Natural killer cells and pregnancy. Nat. Rev. Immunol. 2, 656–663. 4. Hawgood S, Poulain FR 2001 The pulmonary collectins and surfactant metabolism. Annu Rev Physiol 63:495–519. 5. Madsen, J., I. Tornoe, O. Nielsen, C. Koch, W. Steinhilber, and U. Holmskov. 2003. Expression and localization of lung surfactant protein A in human tissues. Am. J. Respir. Cell Mol. Biol. 29: 591– 597. 6. Condon JC Jeyasuria P Faust JM Mendelson CR 2004 Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition. Proc Natl Acad Sci USA 101:4978 – 4983 .
