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Synthetic PreImplantation Factor (PIF) prevents fetal loss by modulating LPS induced inflammatory response.

ABSTRACT: Maternal control of inflammation is essential during pregnancy and an exaggerated response is one of the underlying causes of fetal loss. Inflammatory response is mediated by multiple factors and Toll-like receptors (TLRs) are central. Activation of TLRs results in NALP-3 mediated assembly of apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 into the inflammasome and production of pro-inflammatory cytokines IL-1? and IL-18. Given that preventing measures are lacking, we investigated PreImplantation Factor (PIF) as therapeutic option as PIF modulates Inflammation in pregnancy. Additionally, synthetic PIF (PIF analog) protects against multiple immune disorders. We used a LPS induced murine model of fetal loss and synthetic PIF reduced this fetal loss and increased the embryo weight significantly. We detected increased PIF expression in the placentae after LPS insult. The LPS induced serum and placenta cytokines were abolished by synthetic PIF treatment and importantly synthetic PIF modulated key members of inflammasome complex NALP-3, ASC, and caspase-1 as well. In conclusion our results indicate that synthetic PIF protects against LPS induced fetal loss, likely through modulation of inflammatory response especially the inflammasome complex. Given that synthetic PIF is currently tested in autoimmune diseases of non-pregnant subjects (clinicaltrials.gov, NCT02239562), therapeutic approach during pregnancy can be envisioned.

SUBMITTER: Di Simone N

PROVIDER: S-EPMC5507516 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Synthetic PreImplantation Factor (PIF) prevents fetal loss by modulating LPS induced inflammatory response.

Di Simone Nicoletta N Di Nicuolo Fiorella F Marana Riccardo R Castellani Roberta R Ria Francesco F Veglia Manuela M Scambia Giovanni G Surbek Daniel D Barnea Eytan E Mueller Martin M

PloS one 20170712 7

Maternal control of inflammation is essential during pregnancy and an exaggerated response is one of the underlying causes of fetal loss. Inflammatory response is mediated by multiple factors and Toll-like receptors (TLRs) are central. Activation of TLRs results in NALP-3 mediated assembly of apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 into the inflammasome and production of pro-inflammatory cytokines IL-1β and IL-18. Given that preventing measures are lacking, ...[more]

PMID: 28704412

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Project description:BackgroundIntimate embryo-maternal interaction is paramount for pregnancy success post-implantation. The embryo follows a specific developmental timeline starting with neural system, dependent on endogenous and decidual factors. Beyond altered genetics/epigenetics, post-natal diseases may initiate at prenatal/neonatal, post-natal period, or through a continuum. Preimplantation factor (PIF) secreted by viable embryos promotes implantation and trophoblast invasion. Synthetic PIF reverses neuroinflammation in non-pregnant models. PIF targets embryo proteins that protect against oxidative stress and protein misfolding. We report of PIF's embryotrophic role and potential to prevent developmental disorders by regulating uterine milieu at implantation and first trimester.MethodsPIF's effect on human implantation (human endometrial stromal cells (HESC)) and first-trimester decidua cultures (FTDC) was examined, by global gene expression (Affymetrix), disease-biomarkers ranking (GeneGo), neuro-specific genes (Ingenuity) and proteins (mass-spectrometry). PIF co-cultured epidermal growth factor (EGF) in both HESC and FTDC (Affymetrix) was evaluated.ResultsIn HESC, PIF promotes neural differentiation and transmission genes (TLX2, EPHA10) while inhibiting retinoic acid receptor gene, which arrests growth. PIF promotes axon guidance and downregulates EGF-dependent neuroregulin signaling. In FTDC, PIF promotes bone morphogenetic protein pathway (SMAD1, 53-fold) and axonal guidance genes (EPH5) while inhibiting PPP2R2C, negative cell-growth regulator, involved in Alzheimer's and amyotrophic lateral sclerosis. In HESC, PIF affects angiotensin via beta-arrestin, transforming growth factor-beta (TGF-?), notch, BMP, and wingless-int (WNT) signaling pathways that promote neurogenesis involved in childhood neurodevelopmental diseases-autism and also affected epithelial-mesenchymal transition involved in neuromuscular disorders. In FTDC, PIF upregulates neural development and hormone signaling, while downregulating genes protecting against xenobiotic response leading to connective tissue disorders. In both HESC and FTDC, PIF affects neural development and transmission pathways. In HESC interactome, PIF promotes FUS gene, which controls genome integrity, while in FTDC, PIF upregulates STAT3 critical transcription signal. EGF abolished PIF's effect on HESC, decreasing metalloproteinase and prolactin receptor genes, thereby interfering with decidualization, while in FTDC, EGF co-cultured with PIF reduced ZHX2, gene that regulates neural AFP secretion.ConclusionsPIF promotes decidual trophic genes and proteins to regulate neural development. By regulating the uterine milieu, PIF may decrease embryo vulnerability to post-natal neurodevelopmental disorders. Examination of PIF-based intervention strategies used during embryogenesis to improve pregnancy prognosis and reduce post-natal vulnerability is clearly in order.

Project description:BackgroundPreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo.MethodsDetermine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy.ResultsPIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control).ConclusionsPIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.

Project description:BackgroundEarly identification of viable pregnancy is paramount for successful reproduction. Detection of specific signals from pre-implantation viable embryos in normal pregnancy circulation would indicate initiation of embryo-maternal interaction and create a continuum to accurately reflect embryo/fetal well-being post-implantation. Viable mammalian embryos secrete PreImplantation Factor (PIF), a biomarker which plays key, multi-targeted roles to promote implantation, trophoblast invasion and modulate maternal innate and adaptive immunity toward acceptance. Anti-PIF monoclonal antibody (mAb-based chemiluminescent ELISA) accurately detects PIF in singly cultured embryos media and its increased levels correlate with embryo development up to the blastocyst stage. Herein reported that PIF levels (ELISA) in early maternal serum correlate with pregnancy outcome.MethodsArtificially inseminated (AI) blind-coded Angus cattle (N = 21-23) serum samples (day 10,15 & 20 post-AI) with known calf birth were blindly tested, using both non-pregnant heifers (N = 30) and steer serum as negative controls. Assay properties and anti-PIF monoclonal antibody specificity were determined by examining linearity, spike and recovery experiments and testing the antibody against 234 different circulating proteins by microarray. Endogenous PIF was detected using <3 kDa filter separation followed by anti-PIF mAb-based affinity chromatography and confirmed by ELISA and HPLC. PIF expression was established in placenta using anti-PIF mAb-based IHC.ResultsPIF detects viable pregnancy at day 10 post-AI with 91.3% sensitivity, reaching 100% by day 20 and correlating with live calf birth. All non-pregnant samples were PIF negative. PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera. Assay is linear and spike and recovery data demonstrates lack of serum interference. Anti-PIF mAb is specific and does not interact with circulating proteins. Anti-PIF based affinity purification demonstrates that endogenous PIF is what ELISA detects. The early bovine placenta expresses PIF in the trophoblast layer.ConclusionData herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation. PIF ELISA emerges as practical tool to detect viable early pregnancy from day 20 post-AI.

Project description:Abnormal bone metabolism and subsequence osteoporotic fractures are common complications of chronic inflammatory diseases. No effective treatment for these bone-related complications is available at present. The chronic inflammatory state in these diseases has been considered as a key factor of bone loss. Therefore, the combination of inflammation inhibition and bone loss suppression may be an important strategy for reducing bone damage associated with inflammatory diseases. Bushen Huoxue Decoction (BSHXD) is a traditional Chinese herbal compound that has demonstrated the ability to improve bone quality and increase bone density. However, the efficacy of BSHXD on inflammatory bone loss and its underlying mechanisms remain unclear. This study aimed to investigate whether BSHXD inhibits inflammatory bone loss in mice and its potential molecular mechanisms. In the present study, the effect of BSHXD on lipopolysaccharide (LPS)-induced M1 polarization of RAW264.7 macrophage and on local inflammatory bone loss model of mouse skull was determined. The results showed that after treating RAW264.7 cells with LPS for 24 h, the expression levels of IL-1β (39.42 ± 3.076 ng/L, p < 0.05), IL-6 (49.24 ± 1.766 mg/L, p < 0.05) and TNF-α (286.3 ± 27.12 ng/L, p < 0.05) were significantly increased. The addition of BSHXD decreased the expression levels of IL-1β, IL-6, and TNF-α to 31.55 ± 1.296 ng/L, 37.94 ± 0.8869 mg/L, and 196.4 ± 25.25 ng/L, respectively (p < 0.05). The results of immunofluorescence staining, Western blotting (WB) and flow cytometry indicated that the proportion of M1 macrophages in RAW264.7 cells treated with BSHXD for 24 h was significantly lower than that in the LPS group (13.36% ± 0.9829% VS 24.80% ± 4.619%, p < 0.05). The evidence from in-vitro experiments showed that the immunomodulatory ability of BSHXD may be associated with the activation of AMP-dependent protein kinase (AMPK) pathway in LPS-treated macrophages. In addition, the results of micro-CT, H&E staining, immunohistochemical staining and immunofluorescence staining of mouse skull further demonstrated that BSHXD treatment significantly alleviated LPS-induced local bone loss and inflammatory damage in mouse skull model. All results indicated that BSHXD significantly inhibited inflammatory factors release and M1 polarization of macrophage through AMPK signaling pathway. Therefore, BSHXD may be a promising drug for the treatment of inflammatory bone loss.

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Synthetic PreImplantation Factor (PIF) prevents fetal loss by modulating LPS induced inflammatory response.

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Synthetic PreImplantation Factor (PIF) prevents fetal loss by modulating LPS induced inflammatory response.

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