Generation of sgp130Fc transgenic mice: Inhibition of IL-6-transsignaling in vivo impairs the proper resolution of acute inflammation

Interleukin 6 (IL6) cytokine signaling is based on classic and trans-signaling. Classic signaling utilizes the membrane bound Interleukin 6 receptor (IL6R), whereas in trans-signaling IL6 acts via a soluble form of the IL6 receptor. The signal transducing subunit gp130 of the IL6 receptor complex is ubiquitously expressed whereas the IL6R is only sparely expressed. The specific occurrence of the soluble IL6R (sIL6R) during acute and chronic inflammation massively increases the number of target cells for IL6. To address the question whether IL6 trans-signaling is important in vivo transgenic mice were generated, which overexpress the soluble designer receptor antagonist sgp130Fc. To achieve high level accumulation of the sgp130Fc protein in vivo a cDNA-optimization strategy (opt_sgp130Fc) was applied. These mice displayed an IL6-trans-signaling knock out phenotype, since sgp130Fc specifically inhibits only IL6 trans-signaling but not classic IL6 signaling. The transgenic mice are resistant to the IL6/sIL6R-mediated induction of acute phase response. Furthermore, it was shown that during acute inflammation the recruitment of mononuclear cells is strictly dependent on IL6 trans-signaling. The soluble IL6 receptor is released from activated neutrophils at the site of inflammation and in complex with IL6 induces a chemokine expression pattern, which leads to subsequent infiltration of mononuclear cells, and which is specifically blocked in opt_sgp130Fc transgenics

Generation of sgp130Fc transgenic mice: Inhibition of IL-6-transsignaling in vivo impairs the proper resolution of acute inflammation

Interleukin 6 (IL6) cytokine signaling is based on classic and trans-signaling. Classic signaling utilizes the membrane bound Interleukin 6 receptor (IL6R), whereas in trans-signaling IL6 acts via a soluble form of the IL6 receptor. The signal transducing subunit gp130 of the IL6 receptor complex is ubiquitously expressed whereas the IL6R is only sparely expressed. The specific occurrence of the soluble IL6R (sIL6R) during acute and chronic inflammation massively increases the number of target cells for IL6. To address the question whether IL6 trans-signaling is important in vivo transgenic mice were generated, which overexpress the soluble designer receptor antagonist sgp130Fc. To achieve high level accumulation of the sgp130Fc protein in vivo a cDNA-optimization strategy (opt_sgp130Fc) was applied. These mice displayed an IL6-trans-signaling knock out phenotype, since sgp130Fc specifically inhibits only IL6 trans-signaling but not classic IL6 signaling. The transgenic mice are resistant to the IL6/sIL6R-mediated induction of acute phase response. Furthermore, it was shown that during acute inflammation the recruitment of mononuclear cells is strictly dependent on IL6 trans-signaling. The soluble IL6 receptor is released from activated neutrophils at the site of inflammation and in complex with IL6 induces a chemokine expression pattern, which leads to subsequent infiltration of mononuclear cells, and which is specifically blocked in opt_sgp130Fc transgenics

IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis–associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI

Mucus detachment by host metalloprotease meprin \beta requires shedding of its inactive pro-form, which is abrogated by the pathogenic protease RgpB

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease

ATG16L1 orchestrates interleukin-22 signaling in the intestinal epithelium via cGAS-STING.

A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling, which is augmented in Atg16l1 ΔIEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo, IL-22 treatment in Atg16l1 ΔIEC and Atg16l1 ΔIEC/Xbp1 ΔIEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22-induced ileal inflammation in Atg16l1 ΔIEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium

Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response

Aicardi–Goutières syndrome (AGS) provides a monogenic model of nucleic acid‐mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of either RNA:DNA hybrid or genome‐embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid‐sensing pathway. Here, we establish Rnaseh2b (A174T/A174T) knock‐in mice as a subclinical model of disease, identifying significant interferon‐stimulated gene (ISG) transcript upregulation that recapitulates the ISG signature seen in AGS patients. The inflammatory response is dependent on the nucleic acid sensor cyclic GMP‐AMP synthase (cGAS) and its adaptor STING and is associated with reduced cellular ribonucleotide excision repair activity and increased DNA damage. This suggests that cGAS/STING is a key nucleic acid‐sensing pathway relevant to AGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood‐onset interferonopathy and adult systemic autoimmune disorders

Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice

BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis

Automated classification of airborne pollen using neural networks

Pollen allergies are considered as a global epidemic nowadays, as they influence more than a quarter of the worldwide population, with this percentage expected to rapidly increase because of ongoing climate change. To date, alerts on high-risk allergenic pollen exposure have been provided only via forecasting models and conventional monitoring methods that are laborious. The aim of this study is to develop and evaluate our own pollen classification model based on deep neural networks. Airborne allergenic pollen have been monitored in Augsburg, Bavaria, Germany, since 2015, using a novel automatic Bio-Aerosol Analyzer (BAA 500, Hund GmbH). The automatic classification system is compared and evaluated against our own, newly developed algorithm. Our model achieves an unweighted average precision of 83.0 % and an unweighted average recall of 77.1 % across 15 classes of pollen taxa. Automatic, real-time information on concentrations of airborne allergenic pollen will significantly contribute to the implementation of timely, personalized management of allergies in the future. It is already clear that new methods and sophisticated models have to be developed so as to successfully switch to novel operational pollen monitoring techniques serving the above need

Sensitivity of Support Vector Machines to Random Feature Selection in Classification of Hyperspectral Data

Abstract—The accuracy of supervised land cover classifications depends on factors such as the chosen classification algorithm, adequate training data, the input data characteristics, and the se-lection of features. Hyperspectral imaging provides more detailed spectral and spatial information on the land cover than other remote sensing resources. Over the past ten years, traditional and formerly widely accepted statistical classification methods have been superseded by more recent machine learning algorithms, e.g., support vector machines (SVMs), or by multiple classifier systems (MCS). This can be explained by limitations of statistical approaches with regard to high-dimensional data, multimodal classes, and often limited availability of training data. In the pre-sented study, MCSs based on SVM and random feature selection (RFS) are applied to explore the potential of a synergetic use of the two concepts. We investigated how the number of selected features and the size of the MCS influence classification accuracy using two hyperspectral data sets, from different environmental settings. In addition, experiments were conducted with a vary-ing number of training samples. Accuracies are compared with regular SVM and random forests. Experimental results clearly demonstrate that the generation of an SVM-based classifier system with RFS significantly improves overall classification accuracy as well as producer’s and user’s accuracies. In addition, the ensemble strategy results in smoother, i.e., more realistic, classification maps than those from stand-alone SVM. Findings from the experiments were successfully transferred onto an additional hyperspectral data set. Index Terms—Classifier ensembles, hyperspectral data, multi-ple classifier systems (MCSs), random feature selection (RFS), support vector machines (SVMs). I