The level of H2 O2 -type oxidative stress regulates virulence of Theileria-transformed leukocytes

International audienceTheileria annulata infects predominantly macro-phages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macro-phages are aggressively invasive, but this viru-lence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their charac-terization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of mono-cytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α acti-vates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes main-tain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H 2O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels conse-quently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence

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

TGFβ promotes low IL10-producing ILC2 with profibrotic ability involved in skin fibrosis in systemic sclerosis

Objective : Innate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin fibrosis within systemic sclerosis (SSc). Methods : Blood samples and skin biopsies from healthy donor or patients with SSc were analysed by immunostaining techniques. The fibrotic role of sorted ILC2 was studied in vitro on dermal fibroblast and further explored by transcriptomic approach. Finally, the efficacy of a new treatment against fibrosis was assessed with a mouse model of SSc. Results : We found that ILC2 numbers were increased in the skin of patients with SSc and correlated with the extent of skin fibrosis. In SSc skin, KLRG1− ILC2 (natural ILC2) were dominating over KLRG1+ ILC2 (inflammatory ILC2). The cytokine transforming growth factor-β (TGFβ), whose activity is increased in SSc, favoured the expansion of KLRG1- ILC2 simultaneously decreasing their production of interleukin 10 (IL10), which regulates negatively collagen production by dermal fibroblasts. TGFβ-stimulated ILC2 also increased myofibroblast differentiation. Thus, human KLRG1- ILC2 had an enhanced profibrotic activity. In a mouse model of SSc, therapeutic intervention-combining pirfenidone with the administration of IL10 was required to reduce the numbers of skin infiltrating ILC2, enhancing their expression of KLRG1 and strongly alleviating skin fibrosis. Conclusion : Our results demonstrate a novel role for natural ILC2 and highlight their inter-relationships with TGFβ and IL10 in the development of skin fibrosis, thereby opening up new therapeutic approaches in SSc

The Effects of Allicin, a Reactive Sulfur Species from Garlic, on a Selection of Mammalian Cell Lines

Garlic (Allium sativum L.) has been used as a spice and medicinal plant since ancient times. Garlic produces the thiol-reactive defence substance, allicin, upon wounding. The effects of allicin on human lung epithelium carcinoma (A549), mouse fibroblast (3T3), human umbilical vein endothelial cell (HUVEC), human colon carcinoma (HT29) and human breast cancer (MCF7) cell lines were tested. To estimate toxic effects of allicin, we used a standard MTT-test (methylthiazoltetrazolium) for cell viability and 3H-thymidine incorporation for cell proliferation. The glutathione pool was measured using monobromobimane and the formation of reactive species was identified using 2′,7′-dichlorofluoresceine-diacetate. The YO-PRO-1 iodide staining procedure was used to estimate apoptosis. Allicin reduced cell viability and cell proliferation in a concentration dependent manner. In the bimane test, it was observed that cells treated with allicin showed reduced fluorescence, suggesting glutathione oxidation. The cell lines tested differed in sensitivity to allicin in regard to viability, cell proliferation and glutathione oxidation. The 3T3 and MCF-7 cells showed a higher proportion of apoptosis compared to the other cell types. These data show that mammalian cell lines differ in their sensitivity and responses to allicin

Data from: Ultrasonic cavitation induces necrosis and impairs growth in three-dimensional models of pancreatic ductal adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a rapidly increasing cause of mortality whose dismal prognosis is mainly due to overwhelming chemoresistance. New therapeutic approaches include physical agents such as ultrasonic cavitation, but clinical applications require further insights in the mechanisms of cytotoxicity. Three dimensional in vitro culture models such as spheroids exploit realistic spatial, biochemical and cellular heterogeneity that may bridge some of the experimental gap between conventional in vitro and in vivo experiments. Purpose: to assess the feasibility and efficiency of inertial cavitation associated or not with chemotherapy, in a spheroid model of PDAC. Methods: we used DT66066 cells ,derived from a genetically-engineered murine PDAC, isolated from KPC-transgenic mice (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1- Cre). Spheroids were obtained by either a standard centrifugation-based method, or by using a magnetic nano-shuttle method allowing the formation of spheroids within 24 hours and facilitating their handling. The spheroids were exposed to ultrasonic inertial cavitation in a specially designed setup. Four conditions were studied: control, gemcitabine alone , US cavitation alone , US cavitation + gemcitabine. Five US inertial cavitation indexes, corresponding to increased US intensities, were evaluated . The effectiveness of treatment was assessed after 24 hours with the following criteria: spheroid size (growth), ratio of phase S cells (proliferation), proportion of cells in apoptosis or necrosis (cellular mortality). These parameters were assessed by quantitative immunofluorescence techniques. Results: The 3D culture model presented excellent reproducibility. Eight or nine spheroids were analyzed for each condition. Cavitation induced a significant decrease in the size of spheroids , an effect significantly correlated to an increasing cavitation index (p < 0.0001). The treatment induced cell death whose predominant mechanism was necrosis (p < 0.0001). There was a tendency to a synergistic effect of US cavitation and gemcitabine at 5μM concentration, however significant in only one of the cavitation indexes used (p = 0. 013). Conclusion: Ultrasonic inertial cavitation induced a significant reduction of tumor growth in a spheroid model of PDAC., with necrosis rather than apoptosis as a Cell dominant mechanism of cell death. More investigations are needed to understand the potential role of inertial cavitation in overcoming chemoresistance

Different Expression of Hypoxic and Angiogenic Factors in Human Endometriotic Lesions

Endometriosis is associated with local angiogenic and hypoxic mechanisms. Indeed, peritoneal fluid of women with endometriosis generates a specific microenvironment to support the growth and development of ectopic endometrial tissues. The association between proangiogenic markers and hypoxic processes in different endometriosis phenotypes was investigated in the present study, analyzing the expression of several genes, related to hypoxic signaling pathway and involved in angiogenic processes, in nonpregnant women with different forms of endometriosis. Samples of ovarian endometrioma (OMA; n = 16) or deep infiltrating endometriosis (DIE; n = 11) were collected, and in addition, control endometrium was collected from healthy women by hysteroscopy. The gene expression of the hypoxia-inducible factors (HIF) 1/2α, protease-activated receptors (PARs) ¼, and vascular endothelial growth factor (VEGF) A was evaluated by quantitative reverse-transcription polymerase chain reaction. Ovarian endometrioma expresses high levels of HIF-1/2α, PAR-1/4, and VEGF-A, while DIE did not show significantly different gene expression compared to endometrium from unaffected women. A positive correlation between the expression of HIF-1/2α and VEGF-A mRNA was observed in OMA. The overall data point out that the heterogeneity of the disease reflects differences in expression levels of genes associated with hypoxia and angiogenesis, suggesting that such conditions may have an active role in the development of the disease

Therapeutic Potential of a Senolytic Approach in a Murine Model of Chronic GVHD

10.3390/biology12050647BIOLOGY-BASEL12