Frédéric Batteux

Transgenic Expression of Fas Ligand on Thyroid Follicular Cells Prevents Autoimmune Thyroiditis

“Immune privilege” is defined as tissue resistance to aggression by specifically activated lymphocytes, and involves the interaction between Fas expressed on infiltrating cells and Fas ligand (FasL) constitutively expressed on the target tissue. To test whether ectopic expression of FasL on thyrocytes could prevent autoimmune aggression of the thyroid by activated lymphoid cells, three lines of transgenic mice expressing low, intermediate, and high levels of functional FasL on thyroid follicular cells were generated. Experimental autoimmune thyroiditis was induced by immunization with mouse thyroglobulin. In all of the experiments, the effects were dependent on the level of FasL expression. Low and intermediate expression had no or only weak preventive effects, respectively, whereas high FasL expression strongly inhibited lymphocytic infiltration of the thyroid. Anti-mouse thyroglobulin-proliferative and cytotoxic T cell responses, as well as autoantibody production, were diminished in transgenic mice expressing high levels of FasL relative to controls. Furthermore, in these latter mice Th1 responses to mouse thyroglobulin were profoundly down-regulated, uncovering a new potential role for FasL in peripheral tolerance to organ-specific Ags. In sum, the prevention of experimental autoimmune thyroiditis by FasL on thyrocytes is dependent on the level of FasL expression.

Transgenic Expression of CD95 Ligand on Thyroid Follicular Cells Confers Immune Privilege upon Thyroid Allografts

Constitutive Fas ligand (FasL) expression by specialized cells in the body participates in the immune privilege status of tissues containing these cells. This property has been used to prevent rejection of allogeneic grafts. Nevertheless, the mechanism responsible for such protection has not been fully elucidated. Unfortunately, grafting of FasL transgenic (TG) tissues has been unsuccessful. We have generated TG mice expressing FasL (soluble + membrane bound) on thyroid follicular cells (TFC), and used them to show that ectopic FasL expression prevents thyroid allograft rejection. FasL expression on TFC led to markedly decreased anti-allogeneic, cytotoxic, and helper T lymphocyte activities. The alloantibody response in TG thyroid recipients was either completely inhibited or switched toward a T2-Ab response. Surprisingly, the beneficial effect of FasL on TG thyroid grafts was abolished by host CD4+ T cell depletion. Host CD8+ T cell depletion improved nontransgenic (NTG), but not TG graft survival. Altogether, our results suggest that FasL-induced tolerance is concomitant with a move away from a T1 type response, and a CD4 T cell-mediated regulation of the allocytotoxic T cell response. These results were dependent upon the level of FasL expression on TFC, in that low expression of FasL led to a less marked effect compared with the effect observed with high expression of FasL. These results provide some insight into the role of FasL in regulating destructive alloimmune responses in the case of whole organ grafting, and they have important implications for the development of FasL-based immunotherapy in organ transplantation.

Expression of Fas ligand improves the effect of IL-4 in collagen-induced arthritis.

The present study was aimed at investigating whether the expression of Fas ligand (FasL) by CHO cells transfected with IL‐4 (CHOu2009/u2009IL‐4) or IL‐10 (CHOu2009/u2009IL‐10) genes would improve the effect of the cytokine. DBAu2009/u20091 mice immunized with type II collagen were treated with suboptimal doses of transfected CHO cells (a single s.u2009u2009c. injection of 2u2004× 105 cells) around onset of arthritis. Severe collagen‐induced arthritis (CIA) developed in the control groups injected with PBS, CHOu2009/u2009β‐galactosidaseu2009/u2009FasL, CHOu2009/u2009IL‐4 or CHOu2009/u2009IL‐10 cells. In contrast, administration of CHOu2009/u2009IL‐4u2009/u2009FasL, but not CHOu2009/u2009IL‐10u2009/u2009FasL, cells significantly reduced the clinical severity and resulted in rapid and sustained suppressive effect. Amelioration of CIA was not due to a prolonged in vivo secretion of IL‐4 since expression of FasL by CHO cells shortened the in vivo survival of the xenogeneic cells. In fact, administration of FasL+ cells was associated with a decreased proportion of Mac1+ neutrophils in the blood and an increased expression of myeloperoxidase at the site of engineered cell engraftment. These findings suggest that the mechanism underlying the beneficial effect of IL‐4 delivered by cells expressing FasL involves the combination of the anti‐inflammatory properties of IL‐4 and the apoptosis of Fas+ Mac1+ granulocytes participating in the pathogenic process.

HIF‐1α induction, proliferation and glycolysis of Theileria‐infected leukocytes

Within 2u2009h of infection by Theileria annulata sporozoites, bovine macrophages display a two‐ to fourfold increase in transcription of hypoxia inducible factor (HIF‐1α). Twenty hours post‐invasion sporozoites develop into multi‐nucleated macroschizonts that transform the infected macrophage into an immortalized, permanently proliferating, hyper‐invasive and disease‐causing leukaemia‐like cell. Once immortalized Theileria‐infected leukocytes can be propagated as cell lines and even though cultivated under normoxic conditions, both infected B cells and macrophages display sustained activation of HIF‐1α. Attenuated macrophages used as live vaccines against tropical theileriosis also display HIF‐1α activation even though they have lost their tumorigenic phenotype. Here, we review data that ascribes HIF‐1α activation to the proliferation status of the infected leukocyte and discuss the possibility that Theileria may have lost its ability to render its host macrophage virulent due to continuous parasite replication in a high Reactive Oxygen Species (ROS) environment. We propose a model where uninfected macrophages have low levels of H2O2 output, whereas virulent‐infected macrophages produce high amounts of H2O2. Further increase in H2O2 output leads to dampening of infected macrophage virulence, a characteristic of disease‐resistant macrophages. At the same time exposure to H2O2 sustains HIF‐1α that induces the switch from mitochondrial oxidative phosphorylation to Warburg glycolysis, a metabolic shift that underpins uncontrolled infected macrophage proliferation. We propose that as macroschizonts develop into merozoites and infected macrophage proliferation arrests, HIF‐1α levels will decrease and glycolysis will switch back from Warburg to oxidative glycolysis. As Theileria infection transforms its host leukocyte into an aggressive leukaemic‐like cell, we propose that manipulating ROS levels, HIF‐1α induction and oxidative over Warburg glycolysis could contribute to improved disease control. Finally, as excess amounts of H2O2 drive virulent Theileria‐infected macrophages towards attenuation it highlights how infection‐induced pathology and redox balance are intimately linked.

Soluble Ligands for the NKG2D Receptor Are Released during Endometriosis and Correlate with Disease Severity

Background Endometriosis is a benign gynaecological disease. Abundant bulk of evidence suggests that patients with endometriosis have an immunity dysfunction that enables ectopic endometrial cells to implant and proliferate. Previous studies show that natural killer cells have a pivotal role in the immune control of endometriosis. Methods and Findings This is a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n= 202) during surgery for benign gynaecological conditions. After complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free controls women were enrolled. Patients with endometriosis were classified according to a surgical classification in three different types of endometriosis: superficial peritoneal endometriosis (SUP), ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE). Peritoneal fluid samples were obtained from all study participants during the surgery in order to detect soluble NKG2D ligands (MICA, MICB and ULBP-2). When samples with undetectable peritoneal fluid levels of MICA, MICB and ULBP-2 were excluded, MICA ratio levels were significantly higher in endometriosis patients than in controls (median, 1.1 pg/mg; range, 0.1–143.5 versus median, 0.6 pg/mg; range, 0.1–3.5; p=0.003). In a similar manner peritoneal fluid MICB levels were also increased in endometriosis-affected patients compared with disease-free women (median, 4.6 pg/mg; range, 1.2–4702 versus median, 3.4 pg/mg; range, 0.7–20.1; p=0.001). According to the surgical classification, peritoneal fluid soluble MICA, MICB and ULBP-2 ratio levels were significantly increased in DIE as compared to controls (p=0.015, p=0.003 and p=0.045 respectively). MICA ratio levels also correlated with dysmenorrhea (r=0.232; p=0.029), total rAFS score (r=0.221; p=0.031) and adhesions rAFS score (r=0.221; p=0.031). Conclusions We demonstrate a significant increase of peritoneal fluid NKG2D ligands in women with endometriosis especially in those cases presenting DIE. This study suggests that NKG2D ligands shedding is a novel pathway in endometriosis complex pathogenesis that impairs NK cell function.

Control of humoral immunity and auto-immunity by the CXCR4/CXCL12 axis in lupus patients following influenza vaccine

BACKGROUNDnCXCR4 is a chemokine receptor with multiple effects on the immune system, upregulated in patients with SLE, and correlated with disease severity.nnnOBJECTIVEnThis study has investigated whether the levels of CXCR4 expressed on leucocyte subsets in lupus patients are correlated with the efficacy and the safety of the influenza vaccine.nnnMETHODSnTwenty-seven patients were vaccinated and vaccine immunogenicity and tolerance were evaluated. CXCR4 was assayed on leucocyte subsets and correlated with clinical and immunological signs of diseases activity.nnnRESULTSnA significant increase in the titres of antibodies to the three viral strains was observed along with trends towards an increased vaccine efficacy in patients with quiescent disease vs patients with active disease. Recent flu vaccine history and, to a lesser extent, immunosuppressive treatment may influence vaccine immunogenicity. Influenza immunization was not associated with clinical side-effects or clinical lupus flare but with an increase in rheumatoid factor levels. Our study also confirms the correlation of CXCR4 expression with biological autoimmunity as shown by the correlation between the percentage of CXCR4-positive T cells and the ANA titres at D0, and the reverse correlation between CXCR4 expression and vaccine immunogenicity as demonstrated by the higher percentage of CXCR4-positive T cells at D0 and D30 in non-responders vs responders.nnnCONCLUSIONnAltogether, our study confirms the efficacy and the safety of flu vaccine in SLE patients, highlights the role of CXCR4 as a surrogate marker for autoimmunity in lupus and shows that CXCR4 expression on T cells is predictive of vaccine efficacy in SLE patients.

Induction of experimental autoimmune thyroiditis by heat-denatured porcine thyroglobulin: a Tc1-mediated disease

Recent studies have shown that denatured exogenous antigens can prime cytotoxic T lymphocytes (CTL). To assess the contribution of CTL to experimental autoimmune thyroiditis (EAT), porcine thyroglobulin (pTg) was heat‐denatured (hdpTg) and injected i.u2009u2009v. into CBA/J mice, without the aid of adjuvants. Both lymphocytic infiltrations of the thyroid glands and levels of Tg‐specific CTL were similar to those found in conventional EAT induced by Tg and adjuvants. In contrast, proliferative responses could not be detected, and titers of antibodies to pTg were 20 times lower. These EAT‐inducer CTL belong to the CD8+ cell subset and exerted their thyroiditogenic potential through release of IFN‐γ. We conclude that hdpTg‐induced EAT is mediated by type 1 cytotoxic T cells (Tc1).

Dysregulation of the ADAM17/Notch signalling pathways in endometriosis: from oxidative stress to fibrosis

STUDY QUESTIONnIs oxidative stress associated with the A disintegrin and metalloproteases (ADAM) metallopeptidase domain 17 (ADAM17)/Notch signalling pathway and fibrosis in the development of endometriosis?nnnSUMMARY ANSWERnOxidative stress is correlated with hyperactivation of the ADAM17/Notch signalling pathway and a consequent increase in fibrosis in patients with endometriosis.nnnWHAT IS KNOWN ALREADYnIt is nowadays accepted that oxidative stress plays an important role in the onset and progression of endometriosis. Oxidative stress is able to induce the synthesis of some members of the ADAM family, such as ADAM17. ADAM17/Notch signalling is dysregulated in other profibrotic and inflammatory diseases.nnnSTUDY DESIGN, SIZE, DURATIONnThis was a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n = 202) during surgery for a benign gynaecological condition.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnAfter complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free control women were enrolled. Peritoneal fluid (PF) samples were obtained from all the study participants during surgery in order to detect advanced oxidation protein products (AOPPs) and metalloproteinase activity of ADAM17. Stromal cells from endometrial specimens (n = 8) were obtained from endometrium of control patients (Cs), and from eutopic (Es) and ectopic (Ps) endometrium of patients with deep infiltrating endometriosis (DIE) (n = 8). ADAM17, Notch and the fibrosis markers α-smooth muscle actin (α-SMA) and type-I collagen were assessed using immunoblotting in all the endometrial samples obtained. Additionally, fibrosis was assessed after using Notch cleavage inhibitors (DAPT and FLI-06). Notch and fibrosis were also evaluated after stimulation of stromal endometrial cells with ADAM17 purified protein, increasing concentrations of H2O2 and primary cell culture supernatants.nnnMAIN RESULTS AND THE ROLE OF CHANCEnPatients with DIE presented higher PF AOPP and ADAM17 protein levels than controls (P < 0.01 and P < 0.05, respectively). In addition, these two markers were positively correlated (r = 0.614; P < 0.001). At the cellular level, ADAM17 activity was increased in Es and Ps compared to Cs (P < 0.001 and P < 0.01, respectively). Furthermore, Ps presented hyperactivation of Notch signalling (P < 0.05) and augmentation of fibrosis markers (P = 0.009 for α-SMA and P = 0.015 for type-I collagen) compared to controls. The use of DAPT and FLI-06 reduced both fibrosis markers in Ps but not in Cs. Stimulation with ADAM17, H2O2 and Ps supernatant culture significantly increased Notch and fibrosis in both Ps and Cs.nnnLARGE SCALE DATAnN/A.nnnLIMITATIONS REASONS FOR CAUTIONnThe control group consisted of women who underwent surgery for benign gynaecological conditions, which could lead to biases because some of these conditions may cause alterations in oxidative stress and the ADAM17/Notch pathways. The small sample size of endometrial biopsies used for each group of patients (n = 8) is a limitation of the study, and results should be interpreted with caution.nnnWIDER IMPLICATIONS OF THE FINDINGSnWe propose a novel pathway in endometriosis pathogenesis that correlates oxidative stress, hyperactivation of ADAM17/Notch signalling and a consequent increase in fibrosis. This study suggests that Notch signalling plays a key role in the fibrotic processes that take place in ectopic lesions of patients with DIE, as already observed in other pro-fibrotic diseases.nnnSTUDY FUNDING AND COMPETING INTEREST(S)nThis work was supported by grants from University Paris Descartes, INSERM and Fundación Alfonso Martín Escudero. The authors have no competing interests to declare.

The Nrf2-Antioxidant Response Element Signaling Pathway Controls Fibrosis and Autoimmunity in Scleroderma

Systemic sclerosis (SSc) is an autoimmune disease with fibrosis of the skin and internal organs and vascular alterations. Dysregulations in the oxidant/antioxidant balance are known to be a major factor in the pathogenesis of the disease. Indeed, reactive oxygen species (ROS) trigger neoepitopes leading to a breach of immune tolerance and autoimmune responses, activate fibroblasts to proliferate and to produce excess of type I collagen. ROS also alter endothelial cells leading to vascular dysfunction. Glutathione (GSH) is the most potent antioxidant system in eukaryotic cells. Numerous studies have reported a defect in GSH in SSc animal models and humans, but the origin of this defect remains unknown. The transcription factor NRF2 is a key player in the antioxidant defense, as it can induce the transcription of antioxidant and cytoprotective genes, including GSH, through its interaction with the antioxidant response elements. In this work, we investigated whether NRF2 could be implicated in the pathogenesis of SSc, and if this pathway could represent a new therapeutic target in this orphan disease with no curative medicine. Skin biopsies from 11 patients and 10 controls were harvested, and skin fibroblasts were extracted. Experimental SSc was induced both in BALB/c and in nrf2−/− mice by daily intradermal injections of hypochloric acid. In addition, diseased BALB/c mice were treated with an nrf2 agonist, dimethyl fumarate, or placebo. A drop in nrf2 and target genes mRNA levels was observed in skin fibroblasts of SSc patients compared to controls. Moreover, the nrf2 pathway is also downregulated in skins and lungs of SSc mice. In addition, we observed that nrf2−/− mice have a more severe form of SSc with increased fibrosis and inflammation compared to wild-type SSc mice. Diseased mice treated with the nrf2 agonist dimethyl fumarate (DMF) exhibited reduced fibrosis and immune activation compared to untreated mice. The ex vivo treatment of skin fibroblasts from SSc mice with DMF restores GSH intracellular content, decreases ROS production and cell proliferation. These results suggest that the nrf2 pathway is highly dysregulated in human and SSc mice with deleterious consequences on fibrosis and inflammation and that Nrf2 modulation represents a therapeutic target in SSc.