Neutrophil-derived Activin-A moderates their pro-NETotic activity and attenuates collateral tissue damage caused by Influenza A virus infection.

Background: Pre-neutrophils, while developing in the bone marrow, transcribe the Inhba gene and synthesize Activin-A protein, which they store and release at the earliest stage of their activation in the periphery. However, the role of neutrophil-derived Activin-A is not completely understood. Methods: To address this issue, we developed a neutrophil-specific Activin-A-deficient animal model (S100a8-Cre/Inhba fl/fl mice) and analyzed the immune response to Influenza A virus (IAV) infection. More specifically, evaluation of body weight and lung mechanics, molecular and cellular analyses of bronchoalveolar lavage fluids, flow cytometry and cell sorting of lung cells, as well as histopathological analysis of lung tissues, were performed in PBS-treated and IAV-infected transgenic animals. Results: We found that neutrophil-specific Activin-A deficiency led to exacerbated pulmonary inflammation and widespread hemorrhagic histopathology in the lungs of IAV-infected animals that was associated with an exuberant production of neutrophil extracellular traps (NETs). Moreover, deletion of the Activin-A receptor ALK4/ACVR1B in neutrophils exacerbated IAV-induced pathology as well, suggesting that neutrophils themselves are potential targets of Activin-A-mediated signaling. The pro-NETotic tendency of Activin-A-deficient neutrophils was further verified in the context of thioglycollate-induced peritonitis, a model characterized by robust peritoneal neutrophilia. Of importance, transcriptome analysis of Activin-A-deficient neutrophils revealed alterations consistent with a predisposition for NET release. Conclusion: Collectively, our data demonstrate that Activin-A, secreted by neutrophils upon their activation in the periphery, acts as a feedback mechanism to moderate their pro-NETotic tendency and limit the collateral tissue damage caused by neutrophil excess activation during the inflammatory response.

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring.

Redox imbalance and oxidative stress have emerged as generative causes of the structural and functional degradation of red blood cells (RBC) that happens during their hypothermic storage at blood banks. The aim of the present study was to examine whether the antioxidant enhancement of stored RBC units following uric (UA) and/or ascorbic acid (AA) supplementation can improve their storability as well as post-transfusion phenotypes and recovery by using in vitro and animal models, respectively. For this purpose, 34 leukoreduced CPD/SAGM RBC units were aseptically split in 4 satellite units each. UA, AA or their mixture were added in the three of them, while the fourth was used as control. Hemolysis as well as redox and metabolic parameters were studied in RBC units throughout storage. The addition of antioxidants maintained the quality parameters of stored RBCs, (e.g., hemolysis, calcium homeostasis) and furthermore, shielded them against oxidative defects by boosting extracellular and intracellular (e.g., reduced glutathione; GSH) antioxidant powers. Higher levels of GSH seemed to be obtained through distinct metabolic rewiring in the modified units: methionine-cysteine metabolism in UA samples and glutamine production in the other two groups. Oxidatively-induced hemolysis, reactive oxygen species accumulation and membrane lipid peroxidation were lower in all modifications compared to controls. Moreover, denatured/oxidized Hb binding to the membrane was minor, especially in the AA and mix treatments during middle storage. The treated RBC were able to cope against pro-oxidant triggers when found in a recipient mimicking environment in vitro, and retain control levels of 24h recovery in mice circulation. The currently presented study provides (a) a detailed picture of the effect of UA/AA administration upon stored RBCs and (b) insight into the differential metabolic rewiring when distinct antioxidant "enhancers" are used.

Coordinated activation of TGF-ß and BMP pathways promotes autophagy and limits liver injury after acetaminophen intoxication.

Ligands of the transforming growth factor-ß (TGF-ß) superfamily, including TGF-ßs, activins, and bone morphogenetic proteins (BMPs), have been implicated in hepatic development, homeostasis, and pathophysiology. We explored the mechanisms by which hepatocytes decode and integrate injury-induced signaling from TGF-ßs and activins (TGF-ß/Activin) and BMPs. We mapped the spatiotemporal patterns of pathway activation during liver injury induced by acetaminophen (APAP) in dual reporter mice carrying a fluorescent reporter of TGF-ß/Activin signaling and a fluorescent reporter of BMP signaling. APAP intoxication induced the expression of both reporters in a zone of cells near areas of tissue damage, which showed an increase in autophagy and demarcated the borders between healthy and injured tissues. Inhibition of TGF-ß superfamily signaling by overexpressing the inhibitor Smad7 exacerbated acute liver histopathology but eventually accelerated tissue recovery. Transcriptomic analysis identified autophagy as a process stimulated by TGF-ß1 and BMP4 in hepatocytes, with Trp53inp2, which encodes a rate-limiting factor for autophagy initiation, as the most highly induced autophagy-related gene. Collectively, these findings illustrate the functional interconnectivity of the TGF-ß superfamily signaling system, implicate the coordinated activation of TGF-ß/Activin and BMP pathways in balancing tissue reparatory and regenerative processes upon APAP-induced hepatotoxicity, and highlight opportunities and potential risks associated with targeting this signaling system for treating hepatic diseases.

The Post-Storage Performance of RBCs from Beta-Thalassemia Trait Donors Is Related to Their Storability Profile.

Blood donors with beta-thalassemia traits (ßThal+) have proven to be good "storers", since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored ßThal+ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted ßThal+ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that ßThal+ RBCs present a marginal trend for higher recovery, regardless of the recipient's immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored ßThal+ RBCs. Overall, it seems that the intrinsic physiology of ßThal+ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

Activation of both transforming growth factor-ß and bone morphogenetic protein signalling pathways upon traumatic brain injury restrains pro-inflammatory and boosts tissue reparatory responses of reactive astrocytes and microglia.

Various ligands and receptors of the transforming growth factor-ß superfamily have been found upregulated following traumatic brain injury; however, the role of this signalling system in brain injury pathophysiology is not fully characterized. To address this, we utilized an acute stab wound brain injury model to demonstrate that hallmarks of transforming growth factor-ß superfamily system activation, such as levels of phosphorylated Smads, ligands and target genes for both transforming growth factor-ß and bone morphogenetic protein pathways, were upregulated within injured tissues. Using a bone morphogenetic protein-responsive reporter mouse model, we showed that activation of the bone morphogenetic protein signalling pathway involves primarily astrocytes that demarcate the wound area. Insights regarding the potential role of transforming growth factor-ß superfamily activation in glia cells within the injured tissues were obtained indirectly by treating purified reactive astrocytes and microglia with bone morphogenetic protein-4 or transforming growth factor-ß1 and characterizing changes in their transcriptional profiles. Astrocytes responded to both ligands with considerably overlapping profiles, whereas, microglia responded selectively to transforming growth factor-ß1. Novel pathways, crucial for repair of tissue-injury and blood-brain barrier, such as activation of cholesterol biosynthesis and transport, production of axonal guidance and extracellular matrix components were upregulated by transforming growth factor-ß1 and/or bone morphogenetic protein-4 in astrocytes. Moreover, both ligands in astrocytes and transforming growth factor-ß1 in microglia shifted the phenotype of reactive glia cells towards the anti-inflammatory and tissue reparatory 'A2'-like and 'M0/M2'-like phenotypes, respectively. Increased expression of selected key components of the in vitro modulated pathways and markers of 'A2'-like astrocytes was confirmed within the wound area, suggesting that these processes could also be modulated in situ by the integrated action of transforming growth factor-ß and/or bone morphogenetic protein-mediated signalling. Collectively, our study provides a comprehensive comparative analysis of transforming growth factor-ß superfamily signalling in reactive astrocytes and microglia and points towards a crucial role of both transforming growth factor-ß and bone morphogenetic protein pathways in modulating the inflammatory and brain injury reparatory functions of activated glia cells.

Activin, neutrophils, and inflammation: just coincidence?

During the 26 years that have elapsed since its discovery, activin-A, a member of the transforming growth factor ß super-family originally discovered from its capacity to stimulate follicle-stimulating hormone production by cultured pituitary gonadotropes, has been established as a key regulator of various fundamental biological processes, such as development, homeostasis, inflammation, and tissue remodeling. Deregulated expression of activin-A has been observed in several human diseases characterized by an immuno-inflammatory and/or tissue remodeling component in their pathophysiology. Various cell types have been recognized as sources of activin-A, and plentiful, occasionally contradicting, functions have been described mainly by in vitro studies. Not surprisingly, both harmful and protective roles have been postulated for activin-A in the context of several disorders. Recent findings have further expanded the functional repertoire of this molecule demonstrating that its ectopic overexpression in mouse airways can cause pathology that simulates faithfully human acute respiratory distress syndrome, a disorder characterized by strong involvement of neutrophils. This finding when considered together with the recent discovery that neutrophils constitute an important source of activin-A in vivo and earlier observations of upregulated activin-A expression in diseases characterized by strong activation of neutrophils may collectively imply a more intimate link between activin-A expression and neutrophil reactivity. In this review, we provide an outline of the functional repertoire of activin-A and suggest that this growth factor functions as a guardian of homeostasis, a modulator of immunity and an orchestrator of tissue repair activities. In this context, a relationship between activin-A and neutrophils may be anything but coincidental.

Lack of Fas (APO-1/CD95) gene structural alterations or transcript variant ratio changes in breast cancer.

Fas (APO-1/CD95) is a transmembrane receptor protein involved in cell death signaling. Fas receptor and ligand are both expressed in breast cancer cells, however these cells are resistant to apoptosis. Fas gene mutations were detected in hematological and solid tumors, while overexpression of a soluble Fas isoform in serum was related to cancer stage and prognosis. In this work, direct sequencing of exons 6 and 9 of the Fas gene from 90 patients did not reveal any structural alterations. Moreover, no decrease was found in the ratio of the corresponding mRNA species of transmembrane versus soluble Fas isoforms in 31 breast cancer samples compared to 14 controls. Therefore, inhibition of Fas-mediated apoptosis may not be due to structural alterations in the critical exons 6 and 9 of the Fas gene or a shift of expression towards the soluble Fas isoform, but to other mechanisms operating in breast cancer cells.