Long-term MALT1 inhibition in adult mice without severe systemic autoimmunity

The protease MALT1 is a key regulator of NF-kappa B signaling and a novel therapeutic target in autoimmunity and cancer. Initial enthusiasm supported by preclinical results with MALT1 inhibitors was tempered by studies showing that germline MALT1 protease inactivation in mice results in reduced regulatory T cells and lethal multi-organ inflammation due to expansion of IFN-gamma-producing T cells. However, we show that long-term MALT1 inactivation, starting in adulthood, is not associated with severe systemic inflammation, despite reduced regulatory T cells. In contrast, IL-2-, TNF-, and IFN-gamma-producing CD4(+) T cells were strongly reduced. Limited formation of tertiary lymphoid structures was detectable in lungs and stomach, which did not affect overall health. Our data illustrate that MALT1 inhibition in prenatal or adult life has a different outcome and that long-term MALT1 inhibition in adulthood is not associated with severe side effects

Xenogen-free isolation and culture of human adipose mesenchymal stem cells

Background: Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. Methods: Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flow-cytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. Results: Mechanical isolation is more successful in isolating CD34(+)/CD31(-)/CD45(-)/CD13(+)/CD73(+)/CD146(-) ASCs from lipo-aspirate than isolation via collagenase digestion (p < 0.05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. Conclusion: A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications

Novel strategy for rapid functional in vivo validation of oncogenic drivers in haematological malignancies

In cancer research, it remains challenging to functionally validate putative novel oncogenic drivers and to establish relevant preclinical models for evaluation of novel therapeutic strategies. Here, we describe an optimized and efficient pipeline for the generation of novel conditional overexpression mouse models in which putative oncogenes, along with an eGFP/Luciferase dual reporter, are expressed from the endogenous ROSA26 (R26) promoter. The efficiency of this approach was demonstrated by the generation and validation of novel R26 knock-in (KI) mice that allow conditional overexpression of Jarid2, Runx2, MN1 and a dominant negative allele of ETV6. As proof of concept, we confirm that MN1 overexpression in the hematopoietic lineage is sufficient to drive myeloid leukemia. In addition, we show that T-cell specific activation of MN1 in combination with loss of Pten increases tumour penetrance and stimulates the formation of Lyl1(+) murine T-cell lymphoblastic leukemias or lymphomas (T-ALL/T-LBL). Finally, we demonstrate that these luciferase-positive murine AML and T-ALL/T-LBL cells are transplantable into immunocompromised mice allowing preclinical evaluation of novel antileukemic drugs in vivo

Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock

Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability in vivo. In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality

The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice

Background: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk. Objective: We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma. Methods: Ormdl3-LacZ reporter mice, gene-deficient Ormdl3(-/-) mice, and overexpressing Ormdl3(Tg/wt) mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry-based sphingolipidomics were performed, and airway eosinophilia, T(H)2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured. Results: HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3(Tg/wt) mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3(-/-) mice, whereas they were decreased in Ormdl3(Tg/wt) mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wildtype, Ormdl3(Tg/wt), and Ormdl3(-/-) mice across several models of experimental asthma. Conclusion: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma

Tyramide signal amplification for the immunofluorescent staining of ZBP1-dependent phosphorylation of RIPK3 and MLKL after HSV-1 infection in human cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis

A new mouse model to study the role of ectopic Nanos3 expression in cancer

International audienceBACKGROUND:NANOS3 is a gene conserved throughout evolution. Despite the quite low conservation of Nanos sequences between different organisms and even between Nanos paralogs, their role in germ cell development is remarkably universal. Human Nanos3 expression is normally restricted to the gonads and the brain. However, ectopic activation of this gene has been detected in various human cancers. Until now, Nanos3 and other Nanos proteins have been studied almost exclusively in germ cell development.METHODS:Transgenic mice were generated by targeted insertion of a human Nanos3 cDNA into the ROSA26 locus. The transgene could be spatiotemporally induced by Cre recombinase activity removing an upstream floxed STOP cassette. A lung tumor model with ectopic Nanos3 expression was based on the lung-specific activation of the reverse tetracycline transactivator gene, in combination with a tetO-CMV promoter controlling Cre expression. When doxycycline was provided to the mice, Cre was activated leading to deletion of TP53 alleles and activation of both oncogenic KRasG12D and Nanos3. Appropriate controls were foreseen. Tumors and tumor-derived cell cultures were analyzed in various ways.RESULTS:We describe the successful generation of Nanos3LSL/- and Nanos3LSL/LSL mice in which an exogenous human NANOS3 gene can be activated in vivo upon Cre expression. These mice, in combination with different conditional and doxycycline-inducible Cre lines, allow the study of the role of ectopic Nanos3 expression in several cancer types. The Nanos3LSL mice were crossed with a non-small cell lung cancer (NSCLC) mouse model based on conditional expression of oncogenic KRas and homozygous loss of p53. This experiment demonstrated that ectopic expression of Nanos3 in the lungs has a significant negative effect on survival. Enhanced bronchiolar dysplasia was observed when Nanos3-expressing NSCLC mice were compared with control NSCLC mice. An allograft experiment, performed with cell cultures derived from primary lung tumors of control and Nanos3-expressing NSCLC mice, revealed lymph node metastasis in mice injected with Nanos3-expressing NSCLC cells.CONCLUSIONS:A new mouse model was generated allowing examination of Nanos3-associated pathways and investigation of the influence of ectopic Nanos3 expression in various cancer types. This model might identify Nanos3 as an interesting target in cancer therapeutics

Noninvasive whole-body imaging of phosphatidylethanolamine as a cell death marker using 99mTc-duramycin during TNF-induced SIRS

Systemic inflammatory response syndrome (SIRS) is an inflammatory state affecting the whole body. It is associated with the presence of pro- and antiinflammatory cytokines in serum, including tumor necrosis factor (TNF). TNF has multiple effects and leads to cytokine production, leukocyte infiltration, and blood pressure reduction and coagulation, thereby contributing to tissue damage and organ failure. A sterile mouse model of sepsis, TNF-induced SIRS, was used to visualize the temporal and spatial distribution of damage in susceptible tissues during SIRS. For this, a radiopharmaceutical agent, Tc-99m-duramycin, that binds to exposed phosphatidylethanolamine on dying cells was longitudinally visualized using SPECT/CT imaging. Methods: C57BL/6J mice were challenged with intravenous injections of murine TNF or vehicle, and necrostatin-1 was used to interfere with cell death. Two hours after vehicle or TNF treatment, mice received Tc-99m-duramycin intravenously (35.44 +/- 3.80 MBq). Static whole-body Tc-99m-duramycin SPECT/CT imaging was performed 2, 4, and 6 h after tracer injection. Tracer uptake in different organs was quantified by volume-of-interest analysis using PMOD software and expressed as SUVmean. After the last scan, ex vivo biodistribution was performed to validate the SPECT imaging data. Lastly, terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to correlate the obtained results to cell death. Results: An increased Tc-99m-duramycin uptake was detected in mice injected with TNF, when compared with control mice, in lungs (0.55 +/- 0.1 vs. 0.34 +/- 0.05), intestine (0.75 +/- 0.13 vs. 0.56 +/- 0.1), and liver (1.03 +/- 0.14 vs. 0.64 +/- 0.04) 4 h after TNF and remained significantly elevated until 8 h after TNF. The imaging results were consistent with ex vivo gamma-counting results. Significantly increased levels of tissue damage were detected via TUNEL staining in the lungs and intestine of mice injected with TNF. Interestingly, necrostatin-1 pretreatment conferred protection against lethal SIRS and reduced the Tc-99m-duramycin uptake in the lungs 8 h after TNF (SUV, 0.32 +/- 0.1 vs. 0.51 +/- 0.15). Conclusion: This study demonstrated that noninvasive Tc-99m-duramycin SPECT imaging can be used to characterize temporal and spatial kinetics of injury and cell death in susceptible tissues during TNF-induced SIRS, making it useful for global, whole-body assessment of tissue damage during diseases associated with inflammation and injury

Myb drives B-cell neoplasms and myeloid malignancies in vivo

The proto-oncogene MYB encodes the transcription factor c-MYB (cellular MYB, hereafter called MYB), which is often upregulated or aberrantly activated in cancer, including hematological malignancies.1,2 High Myb levels were especially found in acute myeloid leukemia (AML).2-4 Myb was identified initially as a retroviral oncogene (v-Myb) of avian myeloblastosis virus and E26.5,6 These retroviruses are able to transform immature hematopoietic cells in vitro and induce AMLs in chickens7 and mice.8 In leukemia patients, MYB is highly expressed, and in a subset of patients, this is a consequence of translocations, genomic duplications, or somatic mutations that involve the MYB gene itself.9-12 Furthermore, compelling evidence is accumulating that MYB also acts as a dependency factor for the maintenance of most myeloid, T-, and B-cell leukemias.13-15 Overexpression of viral MYB, a truncated form of MYB that lacks its negative regulatory domain, results in the spontaneous formation of T-cell lymphomas in mice.16 However, the in vivo roles of cellular MYB in tumor initiation remain largely unexplored. Here, we show, for the first time, that hematopoietic-specific overexpression of Myb is sufficient to drive B-cell neoplasms and myeloid malignancies in mice. To evaluate whether elevated MYB expression is sufficient to transform cells in vivo, we developed a conditional Myb overexpression (R26-Myb) mouse model (Figure 1A; supplemental Figure 1A,B) using an optimized pipeline for targeting the Rosa26 (R26) locus,17 which previously allowed us to model AML,17 mantle cell lymphoma,18 and immature T-cell leukemia19 in mice. Cre-mediated removal of the floxed stop cassette in mouse embryonic stem cells resulted in a 10-fold increase of Myb transcripts and a threefold upregulation of MYB protein (supplemental Figure 1C,D). R26Myb mice were crossed with Vav-iCre mice20 to enable R26-driven overexpression of Myb and a Firefly luciferase-reporter in the entire hematopoietic system (supplemental Figure 1E). Homozygous R26-Myb mice have a 10-to 15-fold increase in Myb RNA levels in the thymus and bone marrow (BM) (supplemental Figure 1F,G). Of note, we were not able to show increased MYB protein level of 10-week-old thymocytes (data not shown), probably because T-cell progenitors already express high endogenous MYB levels. We monitored an aging cohort of R26-Myb`g/`g; Vav-iCre`g/ 1 (hereafter named MybVav; n 5 21 with 9 males and 12 females) mice (Figure 1A) and found that 7 out of 21 (33%) animals spontaneously developed hematological malignancies (Figure 1B). Detailed necropsy was performed on 4 animals. MybVav mice had mild anemia and showed increased white blood cell and lymphocyte counts in peripheral blood (Figure 1C). In addition, 3 of them displayed splenomegaly (Figure 1D). Detailed flow cytometric analysis revealed that Myb overexpression resulted in the spontaneous development of both B-cell neoplasms and myeloid malignancies (Figure 1E,F). A selection of 1 MybVav tumor per mouse is represented in Figure 1E, while an overview of the immunophenotype of all MybVav tumors per mouse is shown in Figure 1F. A comparison of these MybVav tumors with their Cre-negative littermate controls can be found in supplemental Figure 2. Although cumulative genetic evidence suggests a role for Myb in T-cel

Association of cell death markers with tumor immune cell infiltrates after chemo-radiation in cervical cancer

Irradiation induces distinct cellular responses such as apoptosis, necroptosis, iron-dependent cell death (a feature of ferroptosis), senescence, and mitotic catastrophe. Several of these outcomes are immunostimulatory and may represent a potential for immunogenic type of cell death (ICD) induced by radiotherapy triggering abscopal effects. The purpose of this study is to determine whether intra-tumoral ICD markers can serve as biomarkers for the prediction of patient's outcomes defined as the metastasis status and survival over a 5-year period. Thirty-eight patients with locally advanced cervical cancer, treated with neoadjuvant chemoradiotherapy using cisplatin were included in this study. Pre-treatment tumor biopsy and post-treatment hysterectomy samples were stained for cell death markers and danger associated molecular patterns (DAMPs): cleaved caspase-3 (apoptosis), phosphorylated mixed lineage kinase domain like pseudokinase (pMLKL; necroptosis), glutathione peroxidase 4 (GPX4; ferroptosis) and 4-hydroxy-2-noneal (4-HNE; ferroptosis), high mobility group box 1 (HMGB1) and calreticulin. Although these markers could not predict the patient's outcome in terms of relapse or survival, many significantly correlated with immune cell infiltration. For instance, inducing ferroptosis post-treatment seems to negatively impact immune cell recruitment. Measuring ICD markers could reflect the impact of treatment on the tumor microenvironment with regard to immune cell recruitment and infiltration. One Sentence SummaryCell death readouts during neoadjuvant chemoradiation in cervical cance