Novel 3D in situ visualization of seal heartworm (Acanthocheilonema spirocauda) larvae in the seal louse (Echinophthirius horridus) by X-ray microCT.

The seal heartworm Acanthocheilonema spirocauda (Nematoda: Onchocercidae) parasitizes the heart and pulmonary arteries of various phocid seals of the Northern Hemisphere. Over many decades, potential vectors of this parasite have been discussed, and to this date, the life cycle is not fully known. The seal louse Echinophthirius horridus (Anoplura: Echinophthiriidae) is an obligatory, permanent and haematophagous ectoparasite of phocids that has been hypothesized to function as obligate intermediate host for A. spirocauda. We examined 11 adult E. horridus specimens collected from stranded harbour seals (Phoca vitulina) in rehabilitation at the Sealcentre Pieterburen by X-ray microCT imaging, aiming to illustrate larval A. spirocauda infection sites in situ. In three of these specimens, thread-like larvae were detected in insect organs. Detailed imaging of the most infected louse revealed a total of 54 A. spirocauda larvae located either in fat bodies or the haemocoel. Histological analysis of the same specimen illustrated nematode cross-sections, confirming X-ray microCT data. The current data strongly suggest that E. horridus is a natural intermediate host for A. spirocauda. Moreover, we demonstrate the potential of X-ray microCT-based imaging as a non-destructive method to analyze host-parasite interactions, especially in the neglected field of marine mammal parasitology.

DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis.

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis-specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1-deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.

TYK2 licenses non-canonical inflammasome activation during endotoxemia.

The non-canonical inflammasome is an emerging crucial player in the development of inflammatory and neurodegenerative diseases. It is activated by direct sensing of cytosolic lipopolysaccharide (LPS) by caspase-11 (CASP11), which then induces pyroptosis, an inflammatory form of regulated cell death. Here, we report that tyrosine kinase 2 (TYK2), a cytokine receptor-associated kinase, is a critical upstream regulator of CASP11. Absence of TYK2 or its kinase activity impairs the transcriptional induction of CASP11 in vitro and in vivo and protects mice from LPS-induced lethality. Lack of TYK2 or its enzymatic activity inhibits macrophage pyroptosis and impairs release of mature IL-1ß and IL-18 specifically in response to intracellular LPS. Deletion of TYK2 in myeloid cells reduces LPS-induced IL-1ß and IL-18 production in vivo, highlighting the importance of these cells in the inflammatory response to LPS. In support of our data generated with genetically engineered mice, pharmacological inhibition of TYK2 reduced LPS-induced upregulation of CASP11 in bone marrow-derived macrophages (BMDMs) and of its homolog CASP5 in human macrophages. Our study provides insights into the regulation of CASP11 in vivo and uncovered a novel link between TYK2 activity and CASP11-dependent inflammation.

Application of a Parallelizable Perfusion Bioreactor for Physiologic 3D Cell Culture.

It is crucial but challenging to keep physiologic conditions during the cultivation of 3D cell scaffold constructs for the optimization of 3D cell culture processes. Therefore, we demonstrate the benefits of a recently developed miniaturized perfusion bioreactor together with a specialized incubator system that allows for the cultivation of multiple samples while screening different conditions. Hence, a decellularized bone matrix was tested towards its suitability for 3D osteogenic differentiation under flow perfusion conditions. Subsequently, physiologic shear stress and hydrostatic pressure (HP) conditions were optimized for osteogenic differentiation of human mesenchymal stem cells (MSCs). X-ray computed microtomography and scanning electron microscopy (SEM) revealed a closed cell layer covering the entire matrix. Osteogenic differentiation assessed by alkaline phosphatase activity and SEM was found to be increased in all dynamic conditions. Furthermore, screening of different fluid shear stress (FSS) conditions revealed 1.5 mL/min (equivalent to ∼10 mPa shear stress) to be optimal. However, no distinct effect of HP compared to flow perfusion without HP on osteogenic differentiation was observed. Notably, throughout all experiments, cells cultivated under FSS or HP conditions displayed increased osteogenic differentiation, which underlines the importance of physiologic conditions. In conclusion, the bioreactor system was used for biomaterial testing and to develop and optimize a 3D cell culture process for the osteogenic differentiation of MSCs. Due to its versatility and higher throughput efficiency, we hypothesize that this bioreactor/incubator system will advance the development and optimization of a variety of 3D cell culture processes.

Cdk6 contributes to cytoskeletal stability in erythroid cells.

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-)polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia - and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.

Multi-Functional OCT Enables Longitudinal Study of Retinal Changes in a VLDLR Knockout Mouse Model.

We present a multi-functional optical coherence tomography (OCT) imaging approach to study retinal changes in the very-low-density-lipoprotein-receptor (VLDLR) knockout mouse model with a threefold contrast. In the retinas of VLDLR knockout mice spontaneous retinal-chorodoidal neovascularizations form, having an appearance similar to choroidal and retinal neovascularizations (CNV and RNV) in neovascular age-related macular degeneration (AMD) or retinal angiomatous proliferation (RAP). For this longitudinal study, the mice were imaged every 4 to 6 weeks starting with an age of 4 weeks and following up to the age of 11 months. Significant retinal changes were identified by the multi-functional imaging approach offering a threefold contrast: reflectivity, polarization sensitivity (PS) and motion contrast based OCT angiography (OCTA). By use of this intrinsic contrast, the long-term development of neovascularizations was studied and associated processes, such as the migration of melanin pigments or retinal-choroidal anastomosis, were assessed in vivo. Furthermore, the in vivo imaging results were validated with histological sections at the endpoint of the experiment. Multi-functional OCT proves as a powerful tool for longitudinal retinal studies in preclinical research of ophthalmic diseases. Intrinsic contrast offered by the functional extensions of OCT might help to describe regulative processes in genetic animal models and potentially deepen the understanding of the pathogenesis of retinal diseases such as wet AMD.

A dual role for autophagy in a murine model of lung cancer.

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.

Normal epidermal growth factor receptor signaling is dispensable for bone anabolic effects of parathyroid hormone.

Although the bone anabolic properties of intermittent parathyroid hormone (PTH) have long been employed in the treatment of osteoporosis, the molecular mechanisms behind this action remain largely unknown. Previous studies showed that PTH increases the expression and the activity of epidermal growth factor receptor (EGFR) in osteoblasts, and activation of ERK1/2 by PTH in osteoblasts was demonstrated to induce the proteolytical release of EGFR ligands and EGFR transactivation. However, conclusive evidence for an important role of the EGFR system in mediating the anabolic actions of intermittent PTH on bone in vivo is lacking. Here, we evaluated the effects of intermittent PTH on bone in Waved-5 (Wa5) mice which carry an antimorphic Egfr allele whose product acts as a dominant negative receptor. Heterozygous Wa5 females and control littermates received a subcutaneous injection of PTH (80 µg/kg) or buffer on 5 days per week for 4 weeks. Wa5 mice had slightly lower total bone mineral density (BMD), but normal cancellous bone volume and turnover in the distal femoral metaphysis. The presence of the antimorphic Egfr allele neither influenced the PTH-induced increase in serum osteocalcin nor the increases in distal femoral BMD, cortical thickness, cancellous bone volume, and cancellous bone formation rate. Similarly, the PTH-induced rise in lumbar vertebral BMD was unchanged in Wa5 relative to wild-type mice. Wa5-derived osteoblasts showed considerably lower basal extracellular signal-regulated kinase 1/2 (ERK1/2) activation as compared to control osteoblasts. Whereas activation of ERK1/2 by the EGFR ligand amphiregulin was largely blocked in Wa5 osteoblasts, treatment with PTH induced ERK1/2 activation comparable to that observed in control osteoblasts, relative to baseline levels. Our data indicate that impairment of EGFR signaling does not affect the anabolic action of intermittent PTH on cancellous and cortical bone.