A role for Fgl2 (fibroleukin) in thrombophilic-associated human fetal loss has been indicated by previous studies. Clotting of vascular vessels of the placenta and fetus interferes with adequate blood circulation. The human Fgl2 gene was sequenced with identification of six SNPs, suggesting an association with a population of women suffering from recurrent fetal losses. A small sample size however, prevented precise statistical analyses of this association. In vitro, human endothelial cells (HUVECs) were not found to constitutively express Fgl2, but were shown to up-regulate its expression when challenged with IFN-γ and TNF-α. Interestingly, TNF-α was only shown to induce expression of Fgl2 in HUVECs from male donors and not female donors. A larger case-control study is needed to examine the relationship of Fgl2 with recurrent fetal loss. The role of estrogen in the Th1-induced expression of Fgl2 by HUVECs should also be examined.

Fibrinogen-like protein 2 (FGL2) is a known immunomodulator and prothrombinase, expressed by several subsets of immune cells. This thesis explores its potential role during the establishment of pregnancy, in mice, as well as in trophoblast function and in an immune-mediated subtype of preeclampsia (PE), in humans. We first noticed a marked subfertility in Fgl2 knockout (ko) and Fgl2 overexpressing (tg) colonies, where litters were fewer and smaller. To explain this, we mapped spatiotemporal patterns of FGL2 expression in the female reproductive tract and through the estrous cycle. FGL2 is expressed in the ovarian stroma and theca cell layer, peaking shortly before ovulation. Fgl2 ko and tg mice do not show a defect in natural or induced ovulation. FGL2 is expressed in secretory cells of the oviductal epithelium, and Fgl2 ko mice have reduced fertilization efficiency. Fgl2 tg pups are noticeably small, and we find that a reduced ratio of glycogen cells in the junctional zone of their placenta partly explains this. We next investigated the role of FGL2 in trophoblast function, using BeWo and HTR-8/SVneo cell lines. Inflammatory cytokines increase FGL2 expression in BeWo, and FGL2 overexpression promotes syncytialization. We show that it therefore rescues the deleterious effect of inflammation on syncytium formation. In a large cohort of PE and non-PE human placentas, FGL2 is high in a subtype with immune activation, and low in a canonical, anti-angiogenic subtype. Its expression correlates with incidence of chronic inflammatory histopathological lesions, likely driven by immune rejection gene sets. High FGL2 also associates with a high incidence of fibrin deposition in the placenta. Overall, we conclude that FGL2 is involved in several steps of maternal immune adaptation, both before and after pregnancy. Its absence and excess both contribute to mouse subfertility. In the developing and mature placenta, FGL2 is increased by inflammation in the trophoblast and immune compartment of the mature placenta, as a physiological attempt to re-establish immune equilibrium and protect the ongoing pregnancy.

Background and Aim: CD4+CD25+ regulatory T (Treg) cells are important in the maintenance of self-tolerance, regulation of T cell homeostasis, prevention of allograft rejection and in the modulation of immune responses to pathogens. Several groups have recently reported an increased expression of fgl2 in Treg cells by microarray analysis. FGL2, a member of the fibrinogen-like family, was previously shown to act as an immunomodulator by inhibiting DC maturation and T cell proliferation. Based on these findings, the immunoregulatory role of FGL2 as a novel effector molecule of Treg cells was investigated. Results: In agreement with previous studies, high levels of fgl2 transcripts were detected in Treg cells by real-time PCR. In fgl2-/- mice, an increased number and percentage of Treg cells were found with a greater expression of Foxp3 compared with fgl2+/+ Treg cells; however, the suppressive activity of fgl2-/- Treg cells was significantly impaired. Antibody to FGL2 completely inhibited the activity of fgl2+/+ Treg cells in vitro. Consistent with FGL2 contribution to Treg cell activity, targeted deletion of the gene led to an increased immune reactivity of DC, T cells and B cells, and the manifestation of glomerular autoimmunity in aged fgl2-/- mice. The importance of FGL2 as an effector of Treg cells was also demonstrated in MHV-3-induced fulminant hepatitis. Uninfected susceptible BALB/cJ mice had increased numbers of Treg cells and expression of FGL2 compared to uninfected resistant A/J mice. Following MHV-3 infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased, correlating with an increased percentage of Treg cells. Treatment with anti-FGL2 antibody completely inhibited Treg cell activity and protected susceptible BALB/cJ mice against MHV-3-induced liver injury and mortality. Adoptive transfer of fgl2+/+ Treg cells into resistant fgl2-/- mice increased their mortality following MHV-3 infection. Finally, FGL2 treatment led to prolonged skin graft survival in a murine allotransplant model. The suppressive activity of FGL2 was mediated through binding to the inhibitory FcγRIIB receptor expressed on APCs, resulting in inhibition of DC maturation and induction of B cell apoptosis. Conclusion: These studies indicate that FGL2 plays an important immunoregulatory role as an effector cytokine of Treg cells; targeting FGL2 may provide a novel therapeutic approach for the treatment of patients with viral hepatitis, autoimmunity and transplant rejection.

Background and Aim: CD4+CD25+ regulatory T (Treg) cells are important in the maintenance of self-tolerance, regulation of T cell homeostasis, prevention of allograft rejection and in the modulation of immune responses to pathogens. Several groups have recently reported an increased expression of fgl2 in Treg cells by microarray analysis. FGL2, a member of the fibrinogen-like family, was previously shown to act as an immunomodulator by inhibiting DC maturation and T cell proliferation. Based on these findings, the immunoregulatory role of FGL2 as a novel effector molecule of Treg cells was investigated. Results: In agreement with previous studies, high levels of fgl2 transcripts were detected in Treg cells by real-time PCR. In fgl2-/- mice, an increased number and percentage of Treg cells were found with a greater expression of Foxp3 compared with fgl2+/+ Treg cells; however, the suppressive activity of fgl2-/- Treg cells was significantly impaired. Antibody to FGL2 completely inhibited the activity of fgl2+/+ Treg cells in vitro. Consistent with FGL2 contribution to Treg cell activity, targeted deletion of the gene led to an increased immune reactivity of DC, T cells and B cells, and the manifestation of glomerular autoimmunity in aged fgl2-/- mice. The importance of FGL2 as an effector of Treg cells was also demonstrated in MHV-3-induced fulminant hepatitis. Uninfected susceptible BALB/cJ mice had increased numbers of Treg cells and expression of FGL2 compared to uninfected resistant A/J mice. Following MHV-3 infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased, correlating with an increased percentage of Treg cells. Treatment with anti-FGL2 antibody completely inhibited Treg cell activity and protected susceptible BALB/cJ mice against MHV-3-induced liver injury and mortality. Adoptive transfer of fgl2+/+ Treg cells into resistant fgl2-/- mice increased their mortality following MHV-3 infection. Finally, FGL2 treatment led to prolonged skin graft survival in a murine allotransplant model. The suppressive activity of FGL2 was mediated through binding to the inhibitory FcγRIIB receptor expressed on APCs, resulting in inhibition of DC maturation and induction of B cell apoptosis. Conclusion: These studies indicate that FGL2 plays an important immunoregulatory role as an effector cytokine of Treg cells; targeting FGL2 may provide a novel therapeutic approach for the treatment of patients with viral hepatitis, autoimmunity and transplant rejection.

Chronic viral infection is a significant burden on healthcare systems worldwide. Robust anti-viral immune responses are essential for viral clearance. Persistent viruses use a variety of mechanisms to evade immune surveillance including the upregulation of host immunesuppressive factors. Secreted fibrinogen-like protein 2 (FGL2) has been identified as an inhibitory effector molecule in suppressing immune responses in patients with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) disease. In a murine model of chronic infection caused by Lymphocytic choriomeningitis virus (LCMV) clone 13, we demonstrate that mice deficient in Fgl2 have increased numbers of mature antigen-presenting cells (APC), improved virus-specific cytotoxic T cell immunity and enhanced viral clearance when compared to wild-type mice. These results highlight the importance of the FGL2 inhibitory pathway in immune evasion and provide a rationale to investigate the effects of blocking FGL2 as a novel immune therapeutic in patients suffering from persistent infections.

Chronic viral infection is a significant burden on healthcare systems worldwide. Robust anti-viral immune responses are essential for viral clearance. Persistent viruses use a variety of mechanisms to evade immune surveillance including the upregulation of host immunesuppressive factors. Secreted fibrinogen-like protein 2 (FGL2) has been identified as an inhibitory effector molecule in suppressing immune responses in patients with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) disease. In a murine model of chronic infection caused by Lymphocytic choriomeningitis virus (LCMV) clone 13, we demonstrate that mice deficient in Fgl2 have increased numbers of mature antigen-presenting cells (APC), improved virus-specific cytotoxic T cell immunity and enhanced viral clearance when compared to wild-type mice. These results highlight the importance of the FGL2 inhibitory pathway in immune evasion and provide a rationale to investigate the effects of blocking FGL2 as a novel immune therapeutic in patients suffering from persistent infections.

"Soaring stress hormones hit fertility" (Hohe Spiegel an Stresshormonen mindern die Fertilität) titelt die British Broadcasting Corporation (BBC) online im Juni 2001. Stress ist als schwangerschaftsgefährdendes Element vielfach in klinischen Studien untersucht worden. Gegenstand dieser Arbeit war es, die durch Stress ausgelöste Abortkaskade immunologisch mit anderen Abortstimuli zu vergleichen und zu untersuchen, ob eine gemeinsame abortive "Endstrecke" über die Prothrombinase fibrinogen like protein 2 (fgl2) existiert. In den Versuchen im Mausmodell zeigte sich, dass der pathophysiologische Mechanismus, der zum Abort führt, entscheidend von dem Abort auslösenden Stimulus abhängt. So scheint Stress einen Abort primär über TNF-alpha und Apoptose zu vermitteln. Stark inflammatorische Reize, wie sie bei mikrobiellen Infektionen mit erhöhter Expression von LPS und erhöhten Werte an IL-12 auftreten, induzieren eine Koexpression von TNF-alpha und IFN-gamma. Die Ko-Expression dieser beiden Th1-Zytokinen scheint notwendig zu sein, um die Prothrombinase fgl2 heraufzuregulieren. Die klinischen Daten dieser Arbeit, die an Plazentagewebe von Patientinnen mit Spontanabort beziehungsweise einer Präeklampsie erhoben wurden, zeigen einen deutlichen, lokalen Anstieg des Th1-Zytokin TNF-alpha und eine vermehrte Expression von fgl2 Protein im Gefäßendothel im Vergleich zu Plazentagewebe von unkomplizierten Schwangerschaften. Gleichzeitig konnte eine deutliche Fibrinablagerung beobachtet werden. Eine solche Immunkonstellation könnte durchaus die erfolgreiche Implantation und die ausreichende Ernährung des heranwachsenden Embryos/Fötus einschränken und wichtige, pathophysiologische Grundlage für die Manifestation eines Spontanaborts oder einer Präeklampsie sein. / "Soaring stress hormones hit fertility" is the headline of the British Broadcasting Cooperation online in June 2001. Stress is known to be harmful during pregnancy as many clinical studies demonstrated. The aim of this work was to compare the immunological mechanism of a stress-induced abortion with other abortive stimuli and whether there exists a shared final route via up-regulation of fibrinogen-like protein 2 (fgl2). In the experiments in mice it turned out that the pathophysiological mechanism by which the abortion is mediated depend on the abortive stimuli. It has been demonstrated that stress induces the mRNA expression of TNF-alpha and increases apoptosis. Strong inflammatory stimuli as represented by the injection of IL-12, which is normally upregulated by bacterial LPS, induces the mRNA co-expression of TNF-alpha and IFN-gamma in comparison to normal pregnancies in mice. It seems that this co-expression of TNF-alpha and IFN-gamma is needed for induction of the novel prothrombinase fgl2. The clinical data of this work show an up-regulation of TNF-alpha as well as fgl2 in the pregnancy complications, spontaneous abortion and pre-eclampsia, especially in endothelial cells. This up-regulation was associated with an increase in fibrin deposition. Such an immune constellation might interfere with successful implantation as well as sufficient nutrition of the growing embryo/fetus. This could be one of the pathophysiological trigger for the manifestation of a spontaneous abortion or a pre-eclampsia.

Le cancer colorectal (CCR) est un problème majeur de santé publique. Les nouvelles stratégies thérapeutiques incluant les anticorps anti-EGFR ou anti-VEGF en association avec les chimiothérapies ont amélioré la survie des patients présentant un CCR métastatique. Nous avons évalué le rôle de nouveaux biomarqueurs pour individualiser les patients porteurs d’un CCR métastatique qui peuvent bénéficier d’un traitement par thérapies ciblées. Dans la première partie, nous avons démontré pour la première fois la valeur de pSTAT3 (STAT3 phosphorylé) comme biomarqueur prédictif pour sélectionner les populations porteuses d’un CCR métastatique qui pourraient bénéficier d’un traitement par anti-EGFR. Dans ce cadre nous avons publié les résultats de la valeur prédictive et pronostique de pSTAT3 chez 94 patients porteurs d’un CCR métastatique traités par cétuximab en 2ème ligne de traitement (Dobi et al, 2012).La probabilité d’avoir une réponse objective était de 13% chez les patients avec l’expression de STAT3 comparé à 41% chez les patients pSTAT3 négatif (p=0.02). Nous avons montré également l’impact négatif de pSTAT3 sur la durée de la survie sans progression (PFS) et de la survie globale (OS) des patients présentant un CCR métastatique. Ce résultat montre l’intérêt de STAT3 comme biomarqueur prédictif de traitement par anti-EGFR. Dans la deuxième partie, nous avons évalué l’impact en survie des facteurs d’angiogenèse complexe comme angiopoetin-2, syndecan-1 (CD138), FGL2. Dans ce cadre nous avons étudié une cohorte exploratoire à Besançon de patients porteurs d’un CCR métastatique, traitée par chimiothérapie cytotoxique et bévacizumab (anti-VEGF). Après la détermination des seuils de valeurs de chaque biomarqueurs dans le sérum de patients utilisant les courbes ROC, nous avons évalué l’impact négatif de score élevé d’angiopoetin-2 et CD138 en survie de ces patients. Nous avons également démontré l’impact négatif de la combinaison de 3 marqueurs combinés en termes d’OS et de PFS dans une cohorte de validation incluant 244 patients de Besançon, Tours et Dijon. / Despite the progress made in the management of metastatic colorectal cancer (mCRC), this disease remains a major health problem. Targeted therapies (anti-EGFR and anti-VEGF therapies) have improved the clinical outcomes achieved by conventional chemotherapy regimens. Half of the patients with K-RAS wild type colo-rectal cancer doen’t benefit from adding anti-EGFR to standard chemotherapy regimen. We decided to assess the clinical outcomes of anti-EGFR containing chemotherapy regimens in 94 patients with mCRC according to phosphorylated STAT3 (pSTAT) status. The probability of achieving an objective reponse was 13% among patients with positive nuclear expression of pSTAT3 compared with 41% for patients displaying pSTAT3 négative tumors (p=0.02). In a multivariate logistic regression model, high-grade skin rash, wild-type K-RAS status, and negative pSTAT3 status significantly improved time to progression (TTP) and overal survival (OS). These results underscore the impact of pSTAT3 on the clinical efficacy of anti-EGFR-containing chemotherapy regimens and support the prospective assessment of this biomarker. Inhibition of pSTAT3 in mCRCs might be a promising strategy to target chemotherapy-resistant cancer cells and should be assessed in association with anti-EGFR monoclonal antibodies. The second goal of our study was the identification of angiopoetin-2, syndecan1 (CD 138) and FGL2 as prognostic biomarquers of metastatic colorectal cancer patients. Plasma angiopoetin-2, FGL2 et CD 138 levels were mesured in 10 healthy volunteers, 13 patients with locally advanced CRC et 51 mCRC, received bevacizumab containing chemotherapy. Angiopoetin-2 and CD138 levels were significantly elevated in patients with mCRC compared with healthy controls. Amongst patients with mCRC, low pre-therapeutic plasma angiopoetin-2 and CD 138 levels were associated with a prolonged median progression-free survival (PFS) (p<0.01 and p=0.03 respectively), and overall survival (p<0.01 and p=0.02 respectively). PFS and OS was prolonged in patients with out or with only one detectable biomarker compared with patients with two or three high biomarker levels (p<0.01).