Mise en évidence de dysfonctions liées au développement de l'endométriose péritonéale : contributions angio-inflammatoires des cytokines et prostaglandines

L'endométriose est une maladie gynécologique, touchant les femmes en âge de procréer. Cette pathologie est caractérisée par la présence de tissu endométrial ectopique, c'est-à-dire en dehors de la cavité utérine. Des dysfonctions du système immunitaire sont de plus en plus souvent suspectées comme étant un des éléments responsables de la pathogenèse de cette maladie. L’objectif général de ce projet a donc été d’étudier les mécanismes cellulaires de molécules pro-inflammatoires aux propriétés variées et à l'expression anormalement élevée dans cette pathologie, que sont MIF et les prostaglandines PGE2 et PGF2α, dans les anomalies inflammatoires et invasives en cause dans cette pathologie. La première partie de nos travaux a porté sur l’étude d’un modèle murin de l’endométriose déficient du gène MIF. Le nombre et le volume des lésions collectées à partir des souris déficientes pour le gène MIF sont significativement inférieurs à ceux mesurés dans des souris sauvages utilisées comme contrôle. L’analyse par PCR des cellules isolées des lésions de souris déficientes du gène MIF a révélé une expression réprimée des protéines d’adhésion, d’inflammation et d’angiogenèse. Ces données démontrent pour la première fois que le MIF agit directement sur la croissance et la progression de lésions d’endométriose in vivo. Une partie de nos travaux a porté sur les molécules nécessaires au métabolisme de PGE2 et PGF2α dans l'endomètre eutopique des femmes normales et l'endomètre eutopique et ectopique des femmes atteintes d'endométriose. Selon nos données, l'expression de certains de ces facteurs est perturbée durant cette maladie, ce qui peut avoir des effets délétères sur la physiologie de la procréation. La stimulation des cellules ectopiques par PGF2α entraîne une libération accrue de VEGF et CXCL-8, ceci via l'induction de COX-2 et des deux variants d’épissage du récepteur FP. De plus, la PKC joue un rôle dans ce phénomène, dépendamment et indépendamment de la PLC. Par son effet inducteur sur la libération de VEGF et CXCL-8, PGF2α pourrait favoriser l'aspect inflammatoire et le développement ectopique des lésions d'endométriose, notamment par des phénomènes d’angiogenèse et de prolifération cellulaire accrus. L'effet de PGF2α sur la libération de VEGF et CXCL-8 par les cellules endométriales ectopiques pourrait également expliquer les quantités élevées de ces cytokines dans le liquide péritonéal des femmes atteintes d'endométriose, un phénomène suspecté dans l'infertilité et les douleurs associées à cette maladie. Nos derniers résultats obtenus à partir du liquide péritonéal montrent un profil cytokinique en faveur de l’angiogenèse et la prolifération des lésions d’endométriose, avec une forte augmentation des facteurs suivants : EGF, FGF-2, IL-1α, MIP-1β, TGFα, PDGF-AA, PDGF-BB, MCP-3, sCD40L, Gro Pan, IL-17α, MDC et Rantes, confortant nos observations préalables redéfinissant la maladie comme étant d’origine angio-inflammatoire. L'endométriose et ses symptômes sont des phénomènes complexes ayant probablement plus qu'une seule origine. Parmi les nombreux facteurs à l'expression altérée dans l'endométriose, notre étude montre que MIF, PGE2 et PGF2α, ainsi qu’une pléthore de facteurs pro-angiogéniques pourraient être de ceux jouant un rôle dans l'infertilité et les douleurs reliées à cette maladie.Endometriosis is a menstrual disorders, is mainly diagnosed in the peritoneal cavity by the presence of lesions, which are thought to originate from the endometrium. Little is known about the causes of endometriosis and no targeted treatment is available. Our studies were the first to show functional defects involving the immune system in the endometrium, i.e. before this tissue migrates and grows into abnormal locations. Our current main hypothesis is that endometriosis development requires a combination of immune dysfunction involving factors, such as MIF, PGE2 and PGF2α. Using a mouse model where MIF-knock out (KO) mice received intra-peritoneal injection of endometrial tissue from MIF-KO or syngenic wild type (WT) mice and vice versa, we first revealed that MIF genetic depletion resulted in a marked reduction ectopic endometrial tissue growth, a disrupted tissue structure and a significant downregulation of the expression of major inflammatory, cell adhesion, survival and angiogenic factors relevant to endometriosis pathogenesis, whereas MIF add-back to MIF-KO mice significantly restored endometriosis-like lesions number and size. This study provides compelling evidence for the role of MIF in endometriosis development and its possible interest for a targeted treatment of endometriosis. We then revealed for the first time multiple defects in PG biosynthesis and receptivity pathways, which differ between eutopic intrauterine and ectopic endometrial tissues and may, owing to the wide spectrum of PG properties, contribute to the initial steps of endometrial tissue growth and development and have an important role to play in the pathogenesis and symptoms of this disease. Afterward, we focussed on PGF2α which markedly up-regulated PGE2, CXCL-8 and VEGF secretion in endometriotic cells, through COX-2 activation. Such an effect was abolished by AL8810, a specific FP antagonist, and significantly down-regulated after specific inhibition of FP different variants signalling pathways. PGF2α enhanced angiogenesis through endothelial tubal formation and proliferation processes. These results show for the first time that PGF2α exerts an indirect angiogenic effects by interacting with ectopic stromal cells and induces the secretion of major angiogenic factors via FP signalling pathways. This study provides evidence for a new mechanism underlying endometriosis development and pathophysiology. As our last data showed distinct patterns of peritoneal fluid cytokine concentrations in endometriotic women most notably a marked increase in EGF, FGF-2, IL-1α, MIP-1β, TGFα, PDGF-AA, PDGF-BB, MCP-3, sCD40L, Gro Pan, IL-17α, MDC and Rantes. These changes may exacerbate the local peritoneal angiogenic and proliferative reaction observed in women with endometriosis, and contributes to its pathophysiology. Inflammation is a major hallmark of endometriosis and angiogenesis is crucial to ectopic endometrial tissue growth. Once viewed as archetypical mediators of inflammation and pain, prostaglandins and angiogenic cytokines should be now regarded major promoters of endometriotic lesions growth

Data from: Macrophage migration inhibitory factor is involved in ectopic endometrial tissue growth and peritoneal-endometrial tissue interaction in vivo: a plausible link to endometriosis development

Pelvic inflammation is a hallmark of endometriosis pathogenesis and a major cause of the disease's symptoms. Abnormal immune and inflammatory changes may not only contribute to endometriosis-major symptoms, but also contribute to ectopic endometrial tissue growth and endometriosis development. A major pro-inflammatory factors found elevated in peritoneal fluid of women with endometriosis and to be overexpressed in peritoneal fluid macrophages and active, highly vascularized and early stage endometriotic lesions, macrophage migration inhibitory factor (MIF) appeared to induce angiogenic and inflammatory and estrogen producing phenotypes in endometriotic cells in vitro and to be a possible therapeutic target in vivo. Using a mouse model where MIF-knock out (KO) mice received intra-peritoneal injection of endometrial tissue from MIF-KO or syngeneic wild type (WT) mice and vice versa, our current study revealed that MIF genetic depletion resulted in a marked reduction ectopic endometrial tissue growth, a disrupted tissue structure and a significant down regulation of the expression of major inflammatory (cyclooxygenease-2), cell adhesion (αv and β3 integrins), survival (B-cell lymphoma-2) and angiogenic (vascular endothelial cell growth) factorsrelevant to endometriosis pathogenesis, whereas MIF add-back to MIF-KO mice significantly restored endometriosis-like lesions number and size. Interestingly, cross-experiments revealed that MIF presence in both endometrial and peritoneal host tissues is required for ectopic endometrial tissue growth and pointed to its involvement in endometrial-peritoneal interactions. This study provides compelling evidence for the role of MIF in endometriosis development and its possible interest for a targeted treatment of endometriosis

Immunostaining of PCNA in murine endometrial implants.

<p>PCNA immunostainingwas carried out on endometrial implants from KO/KO mice (A), WT/WT mice (B) and mice treated with ISO-1 (C) or rhMIF (D). Insets show general histological views of endometrial implants. Black arrows show CD74 positive cells. Scale bar,10 µm.</p

Quantitation of total and free teriflunomide (A77 1726) in human plasma by LC-MS/MS

The clinical activity of leflunomide, a drug used in the treatment of rheumatoid arthritis, is due to its active metabolite, teriflunomide. In vitro studies indicate that at least 99% of teriflunomide is expected to be protein bound in human plasma in vivo, leaving <1% in the unbound or ‘free’ state for clinical activity. To examine details of the relationships between leflunomide dosing and patient response, it is necessary to have an assay that is sufficiently sensitive to measure the minor fraction of free teriflunomide in patient samples. Therefore, we aimed to develop and validate an LC–MS/MS method for the measurement of teriflunomide, and use it to determine the total and free teriflunomide concentration in patients with rheumatoid arthritis. Teriflunomide and its deuterated internal standard were extracted from human plasma and separated using a reversed phase method with a C18 column. Detection was conducted with an API 3000 LC–MS/MS System by monitoring selected ions in negative ion MRM. Optimal detection occurred atm/z 269.1/160.0 (teriflunomide) andm/z 273.1/164.0 (teriflunomide-D4). Over a linear range of 5–500 g/L, the inter-batch precision ranged from 1.9 to 8.8% and accuracy from −8.4 to 8.0%. The intra- and inter-batch assay precision for quality control samples ranged from 2.1–5.4% and 5.7–7.1% respectively. The procedure was applied to assess total and free plasma concentrations of teriflunomide in patients with rheumatoid arthritis. Free teriflunomide was approximately 0.11% of total teriflunomide, and there was a significant correlation (r2 = 0.724) between free and total teriflunomide concentrations. A validated, accurate and sensitive method was developed and successfully applied for the measurement of total and free teriflunomide concentration in human plasma samples. This method has been shown to be reproducible and sensitive and can be applied to clinical samples.

Primer sequences for RT-qPCR.

<p>Primer sequences for RT-qPCR.</p

Effect of rhMIF add-back in KO mice or specific inhibition of MIF in WT mice on the development of endometriosis-like lesions.

<p>Wild type (WT) mice; MIF knock out (KO) mice; rhMIF, KOmice treated with rhMIF; ISO-1, WTmice treated with ISO-1; Vehicle, mice treated with PBS instead of rhMIF in KOmice or ISO-1 in WTmice. Data are from 5 mice per group and expressed as mean ± SEM.</p>a<p>p< 0.05 and ^bp< 0.01 compared with the corresponding vehicle-treated group.</p><p>Effect of rhMIF add-back in KO mice or specific inhibition of MIF in WT mice on the development of endometriosis-like lesions.</p

Immunostaining of MIF receptor CD74 in murine endometrial implants.

<p>CD74 immunostaining was carried out on implants from KO/KO mice (A), WT/WT mice (B) and mice treated with ISO-1 (C) or rhMIF (D). Insets show general histological views of endometrial implants. Black arrows show CD74 positive cells. Scale bar, 10 µm.</p

Stereomicroscopic observation of endometriosis-like lesions.

<p>Endometrial implants as labeled with CFDA-SE and observed at sacrifice by fluorescence stereomicroscopy in MIF-KO mice inoculated with endometrial tissue from syngeneic MIF-KO mice (KO/KO) or WT mice inoculated with syngeneic WT murine endometrial tissue (WT/WT) (controls). Endometrial implants in KO/KO mice treated with rhMIF (0.008 mg/kg) or WT/WT mice treated with ISO-1 (4 mg/kg) are shown. Implants were observed by optic light or epi-fluorescence (GFP). Merged images are shown.</p

Real-time PCR analysis of the expression of genes mainly involved in cell adhesion and vascularisation of endometriosis-like lesions.

<p>Histogram representation of the effect of MIF genetic depletion or antagonism <i>versus</i> controls on VEGF (A, B), COX2 (C, D), BCL2 (E, F), BAX (G, H), ITGAV (I, J) and ITGB3 (K, L) mRNA expression in endometriosis-like lesions by quantitative real time PCR. For each factor, the ratio of mRNA level to GAPDH mRNA was determined. Results were from WT and KOmice (n  =  6) with no treatment (controls) and from WT treated with ISO-1 (n  =  5). Data are mean ± SEM; *, p <0.05 and **, p < 0.01 as compared to the control group.</p