TWEAK Appears as a Modulator of Endometrial IL-18 Related Cytotoxic Activity of Uterine Natural Killers

BACKGROUND: TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions. CONCLUSIONS/SIGNIFICANCE: TWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human

Granulocyte-Colony Stimulating Factor related pathways tested on an endometrial ex-vivo model.

INTRODUCTION: Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF) supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF. MATERIALS AND METHODS: Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR. RESULTS: At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G-CSFR), Integrin alpha-V/beta-3 (ITGB3) implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR) described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP) implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control. CONCLUSION: RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its administration in early pregnancy on early embryogenesis still needs to be demonstrated. Nevertheless, rhG-CSF appears as a promising therapy in some difficult and unsolved cases of reproductive failure. Indications of pre-conceptual rhG-CSF supplementation may derive from a diagnosed lack of endometrial expression of some target genes

Evolution of (our) Concepts on Key Determinants of a Successful Pregnancy

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Cytokines and embryo implantation

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Endometrial mRNA expressions variation in control, IF and RM patients.

<p>Figure 1a: G-CSFR mRNA expression variation in control, IF and RM patients. Figure 1b: TYMP mRNA expression variation in control, IF and RM patients. Figure 1c: ITGB3 and PLAUR mRNA expressions variation in control, IF and RM patients. Figure 1d: CD40 mRNA expression variation in control, IF and RM patients. <i>IF: Implantation Failures. RM: Repeated Miscarriages. Results expressed in concentration ratio (Arbitrary Units) between target gene mRNA level and reference gene mRNA level. (*) Statistically Significant Difference, p<0.05</i>.</p

Endometrial mRNA expressions variation after rhG-CSF stimulation.

<p>Figure 2a: G-CSFR mRNA expression variation after rhG-CSF stimulation. Figure 2b: TYMP mRNA expression variation after G-CSF stimulation. Figure 2c: ITGB3 mRNA expression variation after G-CSF stimulation. Figure 2d: PLAUR mRNA expression variation after G-CSF stimulation. <i>Anti G-CSF: 3 days culture with G-CSF blocking antibody daily supplementation at 3 µg/ml. G-CSF (1): 3 days culture with rhG-CSF daily supplementation at 20 ng/ml. G-CSF (2): 3 days culture with rhG-CSF daily supplementation at 100 ng/ml. G-CSF (3): 3 days culture with rhG-CSF daily supplementation at 200 ng/ml. Results expressed in concentration ratio (Arbitrary Units) between target gene mRNA level after culture with and without specific stimulation, each patient being her own control. (*) Statistically Significant Difference, p<0.05</i>.</p