Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

<p>Abstract</p> <p>Background</p> <p>Interferon induced tetratricopeptide repeat protein 2 (IFIT-2, P54) belongs to the type I interferon response genes and is highly induced after stimulation with LPS. The biological function of this protein is so far unclear. Previous studies indicated that IFIT-2 binds to the initiation factor subunit eIF-3c, affects translation initiation and inhibits protein synthesis. The aim of the study was to further characterize the function of IFIT-2.</p> <p>Results</p> <p>Stimulation of RAW264.7 macrophages with LPS or IFN-γ leads to the expression of IFIT-2 in a type I interferon dependent manner. By using stably transfected RAW264.7 macrophages overexpressing IFIT-2 we found that IFIT-2 inhibits selectively LPS induced expression of TNF-α, IL-6, and MIP-2 but not of IFIT-1 or EGR-1. In IFIT-2 overexpressing cells TNF-α mRNA expression was lower after LPS stimulation due to reduced mRNA stability. Further experiments suggest that characteristics of the 3'UTR of transcripts discriminate whether IFIT-2 has a strong impact on protein expression or not.</p> <p>Conclusion</p> <p>Our data suggest that IFIT-2 may affect selectively LPS induced protein expression probably by regulation at different posttranscriptional levels.</p

Investigating APOE, APP-Aβ metabolism genes and Alzheimer’s disease GWAS hits in brain small vessel ischemic disease

Alzheimer’s disease and small vessel ischemic disease frequently co-exist in the aging brain. However, pathogenic links between these 2 disorders are yet to be identified. Therefore we used Taqman genotyping, exome and RNA sequencing to investigate Alzheimer’s disease known pathogenic variants and pathways: APOE ε4 allele, APP-Aβ metabolism and late-onset Alzheimer’s disease main genome-wide association loci (APOE, BIN1, CD33, MS4A6A, CD2AP, PICALM, CLU, CR1, EPHA1, ABCA7) in 96 early-onset small vessel ischemic disease Caucasian patients and 368 elderly neuropathologically proven controls (HEX database) and in a mouse model of cerebral hypoperfusion. Only a minority of patients (29%) carried APOE ε4 allele. We did not detect any pathogenic mutation in APP, PSEN1 and PSEN2 and report a burden of truncating mutations in APP-Aß degradation genes. The single-variant association test identified 3 common variants with a likely protective effect on small vessel ischemic disease (0.54>OR > 0.32, adj. p-value  1, adj. p-val<0.05) together with Apoe, Ms4a cluster and Cd33 during brain hypoperfusion and their overexpression correlated with the ischemic lesion size. Finally, the detection of Aβ oligomers in the hypoperfused hippocampus supported the link between brain ischemia and Alzheimer’s disease pathology

Systemic ablation of MMP9 triggers invasive growth and metastasis of pancreatic cancer via deregulation of IL-6 expression in the bone marrow

Matrix metalloproteinase 9 (MMP9/Gelatinase B) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and plays a central role in tumor cell invasion and metastasis. Here we complemented mechanistic insights in the cancer biology of MMP9 and investigated the effects of specific long-term loss-of-function, by genetic ablation, of MMP9 on PDAC initiation and progression in the well-established KPC mouse model of spontaneous PDAC. Tumor growth and progression were analyzed by histopathology and immunohistochemistry. Invasive growth of PDAC cells was analyzed by both in vitro (proliferation, survival, migration, invasion assays) and in vivo (experimental metastasis assays) methods. Retroviral shRNAi was used to knockdown target genes (MMP9, IL6R). Gene expression was analyzed by qRT-PCR, immunoblot, ELISA, in situ hybridization and zymography. PDAC tumors from MMP9-deficient mice were dramatically larger, more invasive and contained more stroma. Yet, ablation of MMP9 in PDAC cells did not directly promote invasive growth. Interestingly, systemic ablation of MMP9 led to increased IL-6 levels resulting from abrogation of MMP9-dependent SCF-signaling in the bone marrow (BM). IL-6 levels in MMP9-/- mice were sufficient to induce invasive growth and STAT3 activation in PDAC cells via IL-6 receptor (IL6R). Interference with IL6R blocked the increased invasion and metastasis of PDAC cells in MMP9-deficient hosts. In conclusion, ablation of systemic MMP9 initiated fatal communication between maintenance of physiological functions of MMP9 in the BM and invasive growth of PDAC via the IL-6/IL6R/STAT3 axis. Implications: Thus, the beneficial effects of host MMP9 on PDAC are an important caveat for the use of systemic MMP9 inhibitors in cancer

Деякі проблеми використання тимчасово зайнятих земель

<div><p>Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype <em>Yersinia enterocolitica</em> which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different <em>Yersinia</em> mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, <em>Clostridium difficile</em> toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal <em>GILZ</em> promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the <em>GILZ</em> promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of <em>GILZ</em> promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.</p> </div

PHACTR1 genetic variability is not critical in small vessel ischemic disease patients and PcomA recruitment in C57BL/6J mice

Recently, several genome-wide association studies identified PHACTR1 as key locus for five diverse vascular disorders: coronary artery disease, migraine, fibromuscular dysplasia, cervical artery dissection and hypertension. Although these represent significant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL-like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by different degrees of PcomAs patency. We report 3 very rare coding variants in the small vessel ischemic disease-CADASIL-like cohort (p.Glu198Gln, p.Arg204Gly, p.Val251Leu) and a stop-gain mutation (p.Gln273*) in one MCAO mouse. These coding variants do not cluster in PHACTR1 known pathogenic domains and are not likely to play a critical role in small vessel ischemic disease or brain collateral circulation. We also exclude the possibility that copy number variants (CNVs) or a variant enrichment in Phactr1 may be associated with PcomA recruitment in BCCAS mice or linked to diverse vascular traits (cerebral blood flow pre-surgery, PcomA size, leptomeningeal microcollateral length and junction density during brain hypoperfusion) in C57BL/6J mice, respectively. Genetic variability in PHACTR1 is not likely to be a common susceptibility factor influencing small vessel ischemic disease in patients and PcomA recruitment in C57BL/6J mice. Nonetheless, rare variants in PHACTR1 RPEL domains may influence the stroke outcome and are worth investigating in a larger cohort of small vessel ischemic disease patients, different ischemic stroke subtypes and with functional studies

Yersinia enterocolitica Targets Cells of the Innate and Adaptive Immune System by Injection of Yops in a Mouse Infection Model

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80+, 11% of CD11c+, 7% of CD49b+, 5% of Gr1+ cells, 2.3% of CD19+, and 2.6% of CD3+ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19+CD21+CD23+ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops

PHACTR1 genetic variability is not critical in small vessel ischemic disease patients and PcomA recruitment in C57BL/6J mice

Recently, several genome-wide association studies identified PHACTR1 as key locus for five diverse vascular disorders: coronary artery disease, migraine, fibromuscular dysplasia, cervical artery dissection and hypertension. Although these represent significant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL-like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by different degrees of PcomAs patency. We report 3 very rare coding variants in the small vessel ischemic disease-CADASIL-like cohort (p.Glu198Gln, p.Arg204Gly, p.Val251Leu) and a stop-gain mutation (p.Gln273*) in one MCAO mouse. These coding variants do not cluster in PHACTR1 known pathogenic domains and are not likely to play a critical role in small vessel ischemic disease or brain collateral circulation. We also exclude the possibility that copy number variants (CNVs) or a variant enrichment in Phactr1 may be associated with PcomA recruitment in BCCAS mice or linked to diverse vascular traits (cerebral blood flow pre-surgery, PcomA size, leptomeningeal microcollateral length and junction density during brain hypoperfusion) in C57BL/6J mice, respectively. Genetic variability in PHACTR1 is not likely to be a common susceptibility factor influencing small vessel ischemic disease in patients and PcomA recruitment in C57BL/6J mice. Nonetheless, rare variants in PHACTR1 RPEL domains may influence the stroke outcome and are worth investigating in a larger cohort of small vessel ischemic disease patients, different ischemic stroke subtypes and with functional studies.</p

PHACTR1 genetic variability is not critical in small vessel ischemic disease patients and PcomA recruitment in C57BL/6J mice

Recently, several genome-wide association studies identified PHACTR1 as key locus for five diverse vascular disorders: coronary artery disease, migraine, fibromuscular dysplasia, cervical artery dissection and hypertension. Although these represent significant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL-like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by different degrees of PcomAs patency. We report 3 very rare coding variants in the small vessel ischemic disease-CADASIL-like cohort (p.Glu198Gln, p.Arg204Gly, p.Val251Leu) and a stop-gain mutation (p.Gln273*) in one MCAO mouse. These coding variants do not cluster in PHACTR1 known pathogenic domains and are not likely to play a critical role in small vessel ischemic disease or brain collateral circulation. We also exclude the possibility that copy number variants (CNVs) or a variant enrichment in Phactr1 may be associated with PcomA recruitment in BCCAS mice or linked to diverse vascular traits (cerebral blood flow pre-surgery, PcomA size, leptomeningeal microcollateral length and junction density during brain hypoperfusion) in C57BL/6J mice, respectively. Genetic variability in PHACTR1 is not likely to be a common susceptibility factor influencing small vessel ischemic disease in patients and PcomA recruitment in C57BL/6J mice. Nonetheless, rare variants in PHACTR1 RPEL domains may influence the stroke outcome and are worth investigating in a larger cohort of small vessel ischemic disease patients, different ischemic stroke subtypes and with functional studies