PreImplantation Factor (PIF) promotes HLA-G, -E, -F, -C expression in JEG-3 choriocarcinoma cells and endogenous progesterone activity

BACKGROUND: Pregnancy success requires mandatory maternal tolerance of the semi/allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. AIMS: To examine if PIF affects gene expression of human leukocyte antigen (HLA)-G, -E -F and -C in JEG-3 choriocarcinoma cells, and to examine the influence of PIF on local progesterone activity. Methods: PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis. RESULTS: In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, F confirmed also by Western blotting. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1), resulting in improved maternal signalling. CONCLUSION: PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders

Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production

Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\NOD1 and NOD2 crosstalk converged in NF?B activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1? secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1? restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1? secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1? expression, while NOD2 inversely promoted IL-1?. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction

Nalp Signalling is Required in Sertoli Cells for Tight-Junction Protein Interaction

Summary The present study aims to investigate the NALP3 system and its influence on occludin in Sertoli cells, utilising primary murine cells and adult Sertoli cell line as models. Its main goals are the Sertoli cell biology with possible implications on male reproductive functions. Primary and adult Sertoli cells were transfected with NAPL3 siRNA and treated with NOD1 (ie-DAP) and NOD2 (MDP) receptor ligands. There was positive occludin expression levels on transcript (RT-qPCR) and protein (FCS and Immunofluorescence) levels for both cell types. The innate immunity and tight-junction pathways integration serve a protective role for both testis immune barrier and spermatogenesis compartmentalisation maintained by the very same barrier. This integration also points the way for mechanistic research of the disturbances inflicted during an inflammatory response in testis niche.</jats:p

Nalp Signalling is Required in Sertoli Cells for Tight-Junction Protein Interaction

The present study aims to investigate the NALP3 system and its influence on occludin in Sertoli cells, utilising primary murine cells and adult Sertoli cell line as models. Its main goals are the Sertoli cell biology with possible implications on male reproductive functions. Primary and adult Sertoli cells were transfected with NAPL3 siRNA and treated with NOD1 (ie-DAP) and NOD2 (MDP) receptor ligands. There was positive occludin expression levels on transcript (RT-qPCR) and protein (FCS and Immunofluorescence) levels for both cell types. The innate immunity and tight-junction pathways integration serve a protective role for both testis immune barrier and spermatogenesis compartmentalisation maintained by the very same barrier. This integration also points the way for mechanistic research of the disturbances inflicted during an inflammatory response in testis niche

Mir-15A Reconstitution in Prostate Cancer Cell Line Suppresses Cancer Progression Through Down Regulation of MYB and Androgen Receptor Upregulation

Summary Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. MicroRNAs are noncoding RNAs that have a role of post-transcriptional regulators. In this study we investigated how the tumour suppressor miR-15a modulates main transcription factors like cMYB and AR in androgen sensitive prostate cancer cell line LNCaP. The miR-15a inhibitor, mimic, and their negative controls were transfected into LNCaP cells. Real-time PCR analysis was performed in order to estimate the transcript levels of cMYB and AR. Flow cytometry analysis was performed to measure the protein levels of cMYB and AR. A Cell migration assay was done for cells transfected with miR-15a inhibitor and mimic. We found that cMYB is down-regulated and AR is up-regulated by miR-15a on the transcriptional and protein levels. By reconstituting miR-15a, we found that its down regulation in prostate cancer contributes to cMYB-induced cancer progression and reduced androgen receptivity. The ability of miR-15a to suppress cancer cell viability and migration is a very important phenomenon for understanding cancer heterogeneity in regard to adapted therapeutic approach development.</jats:p

Mir-15A Reconstitution in Prostate Cancer Cell Line Suppresses Cancer Progression Through Down Regulation of MYB and Androgen Receptor Upregulation

Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. MicroRNAs are noncoding RNAs that have a role of post-transcriptional regulators. In this study we investigated how the tumour suppressor miR-15a modulates main transcription factors like cMYB and AR in androgen sensitive prostate cancer cell line LNCaP. The miR-15a inhibitor, mimic, and their negative controls were transfected into LNCaP cells. Real-time PCR analysis was performed in order to estimate the transcript levels of cMYB and AR. Flow cytometry analysis was performed to measure the protein levels of cMYB and AR. A Cell migration assay was done for cells transfected with miR-15a inhibitor and mimic. We found that cMYB is down-regulated and AR is up-regulated by miR-15a on the transcriptional and protein levels. By reconstituting miR-15a, we found that its down regulation in prostate cancer contributes to cMYB-induced cancer progression and reduced androgen receptivity. The ability of miR-15a to suppress cancer cell viability and migration is a very important phenomenon for understanding cancer heterogeneity in regard to adapted therapeutic approach development

The Role of microRNA-15A in the Development of Prostate Cancer – Effects on Cell Proliferation and Pro-Inflammatory Signalling

Worldwide prostate cancer is the second leading cause of cancer death among men after lung cancer. MicroRNAs are non-coding, endogenous RNAs and they play a role in tumorigenesis, RNA silencing and post-transcriptional regulation of gene expression. In this study we have investigated microRNA-15a impact on transcription factors cMYB and ETS1 in prostate-carcinoma cell line PC3. The PC3 cells were transfected with a synthetic analogue and inhibitor of microRNA-15a. The study was performed using reverse transcription polymerase chain reaction and flow cytometry methods for assessing the transcript and protein levels of cMYB and ETS1, NFκB stable reporter live cell line. Statistical analysis was performed using One–way ANOVA test. We found that cMYB and ETS1 are up-regulated by the synthetic analogue of microRNA-15a at the transcription and protein level. Transfection with microRNA-15a mimic resulted in NFκB transcription factor activation as found by using the live cell reporter system. There was some opportunistic activity exhibited by the synthetic inhibitor, but less pronounced. Our data suggest that microRNA-15a could participate in prostate cancer progression by modulating cell proliferation and pro-inflammatory signaling and paves a way for further in-depth investigation of the gene regulatory networks underneath