Receptor for advanced glycation endproducts mediates allergic airway inflammation via group 2 innate lymphoid cells

Half of all asthmatics suffer from allergic airway inflammation driven by type 2 immune responses in the lung, yet the molecular mechanisms that control early, initiating events in allergic asthma are poorly understood. Recently, scientists have discovered a new subset of innate cells that can trigger rapid type 2 allergic inflammation, even in the absence of an adaptive immune system. These cells, termed group 2 innate lymphoid cells (ILC2s), are activated by the epithelial alarmin, interleukin (IL)-33, and secrete copious amounts of the type 2 cytokines IL-5 and IL-13 to drive tissue eosinophilia, mucus hypersecretion, and airway hyperresponsiveness (AHR). It is unknown how these cells are recruited to the lung to promote allergic airway inflammation (AAI). The receptor for advanced glycation endproducts (RAGE) is a proinflammatory receptor abundantly expressed in the lung. Previous studies have found that, in the absence of RAGE, IL-5 and IL-13 cytokine responses are impaired, and mouse lungs are completely protected from development of AAI and AHR. It was therefore hypothesized that RAGE was necessary for recruitment of type-2-cytokine-producing ILC2s to the lung during allergen challenge to initiate AAI. The data presented here demonstrate that RAGE is, in fact, necessary for ILC2 accumulation in the lung after allergen challenge. Furthermore, this mechanism appears to be lung-specific and independent of RAGE expression on ILC2s themselves: tissues that do not normally express RAGE (i.e. gastrointestinal tract) mount normal ILC2-driven allergic responses even in RAGE KO mice. The ILC2-activating cytokine, IL-33, was also dependent on RAGE signaling, both upstream to trigger its release from epithelial cells and also downstream to mediate its inflammatory effects. This is the first study examining a parenchymal factor in the recruitment of ILC2s to a specific organ. The exact mechanism by which this is occurring is still under investigation, but it may lead to the discovery of important new therapeutic targets early in the development of AAI

Remote automated multi-generational growth and observation of an animal in low Earth orbit

The ultimate survival of humanity is dependent upon colonization of other planetary bodies. Key challenges to such habitation are (patho)physiologic changes induced by known, and unknown, factors associated with long-duration and distance space exploration. However, we currently lack biological models for detecting and studying these changes. Here, we use a remote automated culture system to successfully grow an animal in low Earth orbit for six months. Our observations, over 12 generations, demonstrate that the multi-cellular soil worm Caenorhabditis elegans develops from egg to adulthood and produces progeny with identical timings in space as on the Earth. Additionally, these animals display normal rates of movement when fully fed, comparable declines in movement when starved, and appropriate growth arrest upon starvation and recovery upon re-feeding. These observations establish C. elegans as a biological model that can be used to detect changes in animal growth, development, reproduction and behaviour in response to environmental conditions during long-duration spaceflight. This experimental system is ready to be incorporated on future, unmanned interplanetary missions and could be used to study cost-effectively the effects of such missions on these biological processes and the efficacy of new life support systems and radiation shielding technologies

Remote automated multi-generational growth and observation of an animal in low Earth orbit

The ultimate survival of humanity is dependent upon colonization of other planetary bodies. Key challenges to such habitation are (patho)physiologic changes induced by known, and unknown, factors associated with long-duration and distance space exploration. However, we currently lack biological models for detecting and studying these changes. Here, we use a remote automated culture system to successfully grow an animal in low Earth orbit for six months. Our observations, over 12 generations, demonstrate that the multi-cellular soil worm Caenorhabditis elegans develops from egg to adulthood and produces progeny with identical timings in space as on the Earth. Additionally, these animals display normal rates of movement when fully fed, comparable declines in movement when starved, and appropriate growth arrest upon starvation and recovery upon re-feeding. These observations establish C. elegans as a biological model that can be used to detect changes in animal growth, development, reproduction and behaviour in response to environmental conditions during long-duration spaceflight. This experimental system is ready to be incorporated on future, unmanned interplanetary missions and could be used to study cost-effectively the effects of such missions on these biological processes and the efficacy of new life support systems and radiation shielding technologies

Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans

It’s a Cell-Eat-Cell World Autophagy and Phagocytosis

The process of cellular eating, or the phagocytic swallowing of one cell by another, is an ancient manifestation of the struggle for life itself. Following the endosymbiotic origin of eukaryotic cells, increased cellular and then multicellular complexity was accompanied by the emergence of autophagic mechanisms for self-digestion. Heterophagy and autophagy function not only to protect the nutritive status of cells, but also as defensive responses against microbial pathogens externally or the ill effects of damaged proteins and organelles within. Because of the key roles played by phagocytosis and autophagy in a wide range of acute and chronic human diseases, pathologists have played similarly key roles in elucidating basic regulatory phases for both processes. Studies in diverse organ systems (including the brain, liver, kidney, lung, and muscle) have defined key roles for these lysosomal pathways in infection control, cell death, inflammation, cancer, neurodegeneration, and mitochondrial homeostasis. The literature reviewed here exemplifies the role of pathology in defining leading-edge questions for continued molecular and pathophysiological investigations into all forms of cellular digestion

The ZJU index is a powerful surrogate marker for NAFLD in severely obese North American women.

IntroductionNon-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the western world and is highly associated with multiple cardiometabolic complications. The Zhejiang University (ZJU) index was first developed to predict NAFLD in Chinese populations, where it was shown to have better predictive value than other currently used indices. The aims of the present study were to 1) determine the diagnostic accuracy of ZJU index in identifying NAFLD in a well-phenotyped cohort of obese middle-aged American women and 2) compare its performance with other non-invasive indices for NAFLD identification.MethodsTo achieve this goal, we performed a retrospective analysis of a prospectively-collected cohort of participants enrolled in a weight loss trial for severe obesity (RENEW, clinicaltrials.gov identifier: NCT00712127). One hundred and seven women between the age of 30 and 55 with obesity class II (BMI 35-39.9 kg/m2) or class III (BMI ≥ 40 kg/m2) were recruited for analyses. Hepatic steatosis was measured using liver/spleen attenuation ratio (L/S ratio) from unenhanced abdominal computed tomography. Beside ZJU index, hepatic steatosis index (HSI), lipid accumulation production index (LAPI), and visceral adiposity index (VAI) were also determined and to compare their performance in predicting NAFLD.ResultsOf 107 obese women in the study, 40 (37.4%) met imaging criteria for NAFLD using cut-off value of L/S ratio ConclusionThe ZJU index is a powerful surrogate marker for NAFLD in severely obese western females and its predictive value was better than that of other commonly used indices for predicting NAFLD. Our study is the first to suggest that the ZJU index could be a promising model for use in western as well as Chinese populations

Acute loss of integrin-based attachment induces general cytosolic protein degradation via a common mechanism.

<p>A) Age synchronised wild-type L1 larvae were grown to young adulthood at 16°C (t = 0 h) before transferring to NGM RNAi plates <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471-Fraser1" target="_blank">[87]</a> seeded with bacteria expressing dsRNA against genes indicated for an additional 72 h (mid-adulthood) at 20°C. The blue stain that appears as circles in the centre of t = 72 h animals is stain in muscles of developing embryos (for example+<i>pat-2</i> and <i>deb-1</i> RNAi). The blue stain that appears as lines in the t = 72 h animals is indicative of <i>lacZ</i> expressing bacteria in the gut (typically near the head, for example+<i>unc-112</i> RNAi). B) <i>dim-1(ra102)</i> mutants were cultured identically to A. C) Wild-type, <i>unc-52^ts</i>, <i>unc-112^ts</i>, <i>unc-112^ts</i>; <i>dim-1(gk54)</i>, and <i>unc-112^ts</i>; <i>dim-1(ra102)</i> animals were age synchronised at L1 stage and grown to young adulthood (t = 0 h) at 16°C, and cultured for an additional 72 h at either 16°C (permissive temperature for the mutation) or 25°C (non-permissive temperature). <i>unc-52^ts</i> and <i>unc-112^ts</i> animals were also cultured under the same conditions in the presence of cycloheximide (+CHx) at 400 µg/ml. In A, B and C approximately 20–30 animals were stained for β-galactosidase activity (blue) at t = 0 h and after 24 h, 48 h (not shown) and 72 h. D) Representative immunoblot analysis of 146-kDa β-galactosidase fusion protein in 30-worm lysates, cultured under the same conditions as in C after temperature-shift to 25°C only. All experiments in A, B, C and D were repeated a minimum of three times. E) Kinetics of loss of β-galactosidase protein from 16°C (t = 0 h) after temperature-shift to 25°C in wild-type (solid line), <i>unc-52^ts</i> (large dashed line) or <i>unc-112^ts</i> (small dashed line) animals. *,**Significant difference between <i>unc-112^ts</i> versus wild-type (P<0.01, P<0.001). †Significant difference between <i>unc-52^ts</i> versus wild-type (P<0.01). F) Kinetics of loss of β-galactosidase protein from 16°C (t = 0 h) after temperature-shift to 25°C in <i>unc-112^ts</i> (small dashed line), <i>unc-112^ts</i>; <i>dim-1(gk54)</i> (large dashed line) or <i>unc-112</i>^ts; <i>dim-1(ra102)</i> (solid line) animals. **Significant difference between <i>unc-112^ts</i> versus <i>unc-112^ts</i>; <i>dim-1(gk54)</i> and <i>unc-112^ts</i>; <i>dim-1(ra102)</i> (P<0.001). Values in E and F are the average of three immunoblots ± SEM. Level of significance in all indicated cases from two way repeated measures ANOVA. Scale bars represent 100 µm.</p

Calpains are important for maintenance of adult <i>C. elegans</i> muscle.

<p>A) Animals expressing a full length translational fusion of <i>gfp</i> to <i>myo-3</i> (myosin heavy chain A) were age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h). Adult animals were then transferred to NGM RNAi plates <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471-Fraser1" target="_blank">[87]</a> seeded with bacteria expressing dsRNA against genes indicated for a further 72 h to mid-adulthood. 20 random animals were picked and scored for identical defects in sarcomere structure in at least two muscles within the animal and this was repeated for 5 independent RNAi treatments (n = 100 animals per condition/time point). Displayed is the percentage of animals where torn or collapsed arrays of sarcomeres were observed (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s005" target="_blank">Figure S5</a>. **Significant difference from control (t = 72 h, (P<0.001)). B) Animals expressing GFP labelled mitochondria and nuclei were grown, treated and analysed as in A with the exception that mitochondrial structure was scored. Displayed is the percentage of animals where moderate fragmentation of the mitochondrial network was observed (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s005" target="_blank">Figure S5</a>. *, **Significant difference from control (t = 72 h, (P<0.01, P<0.001)). C) Animals expressing GFP labelled attachment complexes (UNC-95::GFP) were grown, treated and analysed as in A with the exception that attachment complex structure was scored. Displayed is the percentage of animals where torn or collapsed arrays of sarcomeres were observed were observed (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s005" target="_blank">Figure S5</a>. **Significant difference from control (t = 72 h, (P<0.001)). D) Wild-type, <i>unc-112^ts</i>, and <i>unc-112^ts</i>; <i>dim-1</i>(<i>gk54</i>) were age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h). Adult animals were then transferred to 25°C and grown for a further 72 h to mid-adulthood. Some <i>unc-112^ts</i> animals were also placed on calpain inhibitor II drug plates (5 µg/ml) at t = 0 h and cultured on drug plates for a further 72 h. 30 animals were picked for western blot analysis of DEB-1 levels at t = 0 h, and at 72 h. All experiments were performed at least three times. Displayed are representative western blots for each condition and a graph of the initial DEB-1 remaining at 72 h (average ± SEM for three independent experiments). **Significant difference from all other conditions (P<0.001). All significance values are from two way repeated measures ANOVA.</p