Anja Sterner-Kock

FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation

Intestinal immune homeostasis depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). Epithelial barrier disruption is considered to be a potential cause of inflammatory bowel disease; however, the mechanisms regulating intestinal epithelial integrity are poorly understood. Here we show that mice with IEC-specific knockout of FADD (FADDIEC-KO), an adaptor protein required for death-receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis. Genetic deficiency in RIP3, a critical regulator of programmed necrosis, prevented the development of spontaneous pathology in both the small intestine and colon of FADDIEC-KO mice, demonstrating that intestinal inflammation is triggered by RIP3-dependent death of FADD-deficient IECs. Epithelial-specific inhibition of CYLD, a deubiquitinase that regulates cellular necrosis, prevented colitis development in FADDIEC-KO but not in NEMOIEC-KO mice, showing that different mechanisms mediated death of colonic epithelial cells in these two models. In FADDIEC-KO mice, TNF deficiency ameliorated colon inflammation, whereas MYD88 deficiency and also elimination of the microbiota prevented colon inflammation, indicating that bacteria-mediated Toll-like-receptor signalling drives colitis by inducing the expression of TNF and other cytokines. However, neither CYLD, TNF or MYD88 deficiency nor elimination of the microbiota could prevent Paneth cell loss and enteritis in FADDIEC-KO mice, showing that different mechanisms drive RIP3-dependent necrosis of FADD-deficient IECs in the small and large bowel. Therefore, by inhibiting RIP3-mediated IEC necrosis, FADD preserves epithelial barrier integrity and antibacterial defence, maintains homeostasis and prevents chronic intestinal inflammation. Collectively, these results show that mechanisms preventing RIP3-mediated epithelial cell death are critical for the maintenance of intestinal homeostasis and indicate that programmed necrosis of IECs might be implicated in the pathogenesis of inflammatory bowel disease, in which Paneth cell and barrier defects are thought to contribute to intestinal inflammation.

Identification of early molecular markers for breast cancer

BackgroundThe ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma. Since DCIS is a curable disease, it would be highly desirable to identify molecular markers that allow early detection. Mice transgenic for the WAP-SV40 early genome region were used as a model for DCIS development. Gene expression profiling was carried out on DCIS-bearing mice and control animals. Additionally, a set of human DCIS and invasive mammary tumors were analyzed in a similar fashion. Enhanced expression of these marker genes in human and murine samples was validated by quantitative RT-PCR. Besides, marker gene expression was also validated by immunohistochemistry of human samples. Furthermore in silico analyses using an online microarray database were performed.ResultsIn DCIS-mice seven genes were identified that were significantly up-regulated in DCIS: DEPDC1, NUSAP1, EXO1, RRM2, FOXM1, MUC1 and SPP1. A similar up-regulation of homologues of the murine genes was observed in human DCIS samples. Enhanced expression of these genes in DCIS and IDC (invasive ductal carcinoma) was validated by quantitative RT-PCR and immunohistochemistry.ConclusionsBy comparing murine markers for the ductal carcinoma in situ (DCIS) of the mammary gland with genes up-regulated in human DCIS-samples we were able to identify a set of genes which might allow early detection of DCIS and invasive carcinomas in the future. The similarities between gene expression in DCIS and invasive carcinomas in our data suggest that the early detection and treatment of DCIS is of utmost relevance for the survival of patients who are at high risk of developing breast carcinomas.

Disruption of LTBP-4 function reduces TGF-β activation and enhances BMP-4 signaling in the lung

Disruption of latent TGF-β binding protein (LTBP)–4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-β, whereas secretion of latent TGF-β was significantly increased. Expression and secretion of TGF-β2 and -β3 increased considerably. These results suggested that TGF-β activation but not secretion would be severely impaired in LTBP-4 −/− fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)–4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the −/− fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-β1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4–mediated targeting and activation of TGF-β1 leads to enhanced BMP-4 signaling in mouse lung.

In vivo application of tissue-engineered blood vessels of bacterial cellulose as small arterial substitutes: proof of concept?

BACKGROUND Tissue-engineered blood vessels (TEBVs) represent an innovative approach for overcoming reconstructive problems associated with vascular diseases by providing small-caliber vascular grafts. This study aimed to evaluate a novel biomaterial of bacterially synthesized cellulose (BC) as a potential scaffold for small-diameter TEBV. METHODS Small-diameter blood vessels with a supramolecular fiber network structure consisting of tubular hydrogels from biodesigned cellulose were created using Gluconacetobacter strains and Matrix reservoir technology. BC tubes (length: 100 mm, inner diameter: 4.0-5.0 mm) were applied to replace the carotid arteries of 10 sheep for a period of 3 mo to gain further insights into (a) functional (in vivo) performance, (b) ability of providing a scaffold for the neoformation of a vascular wall and (c) their proinflammatory potential, and the (d) technical feasibility of the procedure. RESULTS Preoperative analysis revealed a bursting strength of the grafts of approximately 800 mm Hg and suture retention strength of 4-5 N. Postexplantation analysis showed a patency rate of 50% (n = 5) and physiological performance of the patent grafts at 4, 8, and 12 wk postoperatively, compared with native arteries. Histologic analysis revealed a neoformation of a vascular wall-like structure along the BC scaffold consisting of immigrated vascular smooth muscle cells and a homogeneous endothelialization of the inner graft surface without signs of prothrombogenic or inflammatory potential. Scanning electron microscopy revealed a confluent luminal endothelial cell layer and the immigration of vascular smooth muscle cells into the BC matrix. CONCLUSIONS BC grafts provide a scaffold for the neoformation of a three-layered vascular wall exhibit attractive properties for their use in future TEBV programs for cardiovascular surgery.

Inactivation of sestrin 2 induces TGF-β signaling and partially rescues pulmonary emphysema in a mouse model of COPD

SUMMARY Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Cigarette smoking has been identified as one of the major risk factors and several predisposing genetic factors have been implicated in the pathogenesis of COPD, including a single nucleotide polymorphism (SNP) in the latent transforming growth factor (TGF)-β binding protein 4 (Ltbp4)-encoding gene. Consistent with this finding, mice with a null mutation of the short splice variant of Ltbp4 (Ltbp4S) develop pulmonary emphysema that is reminiscent of COPD. Here, we report that the mutational inactivation of the antioxidant protein sestrin 2 (sesn2) partially rescues the emphysema phenotype of Ltbp4S mice and is associated with activation of the TGF-β and mammalian target of rapamycin (mTOR) signal transduction pathways. The results suggest that sesn2 could be clinically relevant to patients with COPD who might benefit from antagonists of sestrin function.

Impact of Pump Flow Rate During Selective Cerebral Perfusion on Cerebral Hemodynamics and Metabolism

BACKGROUND Although hypothermic selective cerebral perfusion (SCP) is widely used for cerebral protection during aortic surgery, little is known about the ideal pump-flow management during this procedure. This study explored cerebral hemodynamics and metabolism at two different flow rates. METHODS Fourteen pigs (33 to 38 kg) were cooled on cardiopulmonary bypass to 25°C. After 10 minutes of hypothermic circulatory arrest, the animals were randomly assigned to 60 minutes of SCP at two different pump flow rates: 8 mL·kg(-1)·min(-1) (n = 7) and 18 mL·kg(-1)·min(-1) (n = 7). Microspheres were injected at baseline, coolest temperature, and at 5, 15, 25, and 60 minutes of SCP to calculate cerebral blood flow, cerebral vascular resistance, metabolic rate, and intracranial pressure. RESULTS Cerebral blood flow decreased during cooling to 41% of the baseline value (from 57 ± 10 to 23 ± 4 mL·min(-1)·100 g(-1)). It recovered during the initial 15 minutes of SCP, showing a significantly higher increase (p = 0.017) at high-flow versus low-flow perfusion (139 ± 41 versus 75 ± 22 mL·min(-1)·100 g(-1)). After 60 minutes of SCP the cerebral blood flow almost returned to baseline values in the low-flow group (43 ± 25 mL·min(-1)·100 g(-1)), but showed an unexpected decrease (30 ± 7 mL·min(-1)·100 g(-1)) in the high-flow group. The highest regional cerebral blood flow was seen in the cortex (66 ± 12 mL·min(-1)·100 g(-1)), followed by the cerebellum (63 ± 12 mL·min(-1)·100 g(-1)), the pons (51 ± 17 mL·min(-1)·100 g(-1)), and the hippocampus (36 ± 9 mL·min(-1)·100 g(-1)). Intracranial pressure increased from 11 ± 3 to 13 ± 5 mm Hg during cooling on cardiopulmonary bypass. During low-flow SCP, it stayed stable at baseline values, whereas high-flow perfusion resulted in significantly higher intracranial pressures (17 ± 3 mm Hg; p = 0.001). Changes in cerebral vascular resistance and metabolic rate showed no significant differences between the groups. CONCLUSIONS High-flow SCP provides no benefit during long-term SCP at 25°C. Higher cerebral blood flow during the initial SCP period leads to cerebral edema, with no profit in metabolic rate.

Assessment of in vitro interferon-γ responses from peripheral blood mononuclear cells of cattle infected with Mycoplasma mycoides ssp. mycoides small colony type

Contagious bovine pleuropneumonia (CBPP) is a lung disease caused by the bacterial pathogen Mycoplasma mycoides ssp. mycoides small colony type (MmmSC). It has been spreading due to a number of factors including poor vaccine efficacy and poor sensitivity of current diagnostic tests. The purpose of this study was to assess interferon gamma (IFN-gamma) release after stimulation of peripheral blood mononuclear cells (PBMC) from experimentally infected cattle. PBMC collected from 15 artificially infected animals were incubated with different concentrations of total MmmSC antigen. After 72h of incubation the IFN-gamma release was measured and found to be elevated in 11 animals. We did not observe a correlation between IFN-gamma release of animals with and without pathomorphological gross lesions. Therefore, our data do not confirm a role for CD4 T-lymphocytes in protection, since there is no correlation between IFN-g secretion (supposed to be mainly derived from CD4 T-cells) and disease severity. Additionally, we applied immunocytochemistry on affected lung tissue and detected no build up of T-lymphocytes (CD4 T-cells, CD8 T-cells) but a high presence of myeloid cells.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella

The X‐linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti‐apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase‐mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP‐mediated immune response by inducing the BID‐dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain‐dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

A minor role of CD4+ T lymphocytes in the control of a primary infection of cattle with Mycoplasma mycoides subsp. mycoides.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4+ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known.We investigated the role of CD4+ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4+ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4+ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4+ T cells in control of CBPP.

Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S−/−), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S−/− and Ltbp4-null (Ltbp4−/−) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.