Sites Of Tissue Injury Research Articles

Mesenchymal stem cells overexpressing hepatocyte nuclear factor-4 alpha alleviate liver injury by modulating anti-inflammatory functions in mice

BackgroundMesenchymal stem cells (MSCs) can migrate to tissue injury sites where they can induce multipotential differentiation and anti-inflammation effects to treat tissue injury. When traditional therapeutic methods do not work, MSCs are considered to be one of the best candidates for cell therapy. MSCs have been used for treating several injury- and inflammation-associated diseases, including liver cirrhosis. However, the therapeutic effect of MSCs is limited. In some cases, the anti-inflammatory function of naïve MSCs is not enough to rescue tissue injury.MethodsCarbon tetrachloride (CCl4) was used to establish a mouse liver cirrhosis model. Enhanced green fluorescence protein (EGFP) and hepatocyte nuclear factor-4α (HNF-4α) overexpression adenoviruses were used to modify MSCs. Three weeks after liver injury induction, mice were injected with bone marrow MSCs via their tail vein. The mice were then sacrificed 3 weeks after MSC injection. Liver injury was evaluated by measuring glutamic-pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST) levels. Histological and molecular evaluations were performed to study the mechanisms.ResultsWe found that HNF-4α-overexpressing MSCs had a better treatment effect than unmodified MSCs on liver cirrhosis. In the CCl4-induced mouse liver injury model, we found that HNF-4α-MSCs reduced inflammation in the liver and alleviated liver injury. In addition, we found that HNF-4α promoted the anti-inflammatory effect of MSCs by enhancing nitric oxide synthase (iNOS) expression, which was dependent on the nuclear factor kappa B (NF-κB) signalling pathway.ConclusionsMSCs overexpressing HNF-4α exerted good therapeutic effects against mouse liver cirrhosis due to an enhanced anti-inflammatory effect. Gene modification is likely a promising method for improving the effects of cell therapy.

MTORC1 amplifies the ATF4-dependent de novo serine-glycine pathway to supply glycine during TGF-β1-induced collagen biosynthesis.

The differentiation of fibroblasts into a transient population of highly activated, extracellular matrix (ECM)-producing myofibroblasts at sites of tissue injury is critical for normal tissue repair. Excessive myofibroblast accumulation and persistence, often as a result of a failure to undergo apoptosis when tissue repair is complete, lead to pathological fibrosis and are also features of the stromal response in cancer. Myofibroblast differentiation is accompanied by changes in cellular metabolism, including increased glycolysis, to meet the biosynthetic demands of enhanced ECM production. Here, we showed that transforming growth factor-β1 (TGF-β1), the key pro-fibrotic cytokine implicated in multiple fibrotic conditions, increased the production of activating transcription factor 4 (ATF4), the transcriptional master regulator of amino acid metabolism, to supply glucose-derived glycine to meet the amino acid requirements associated with enhanced collagen production in response to myofibroblast differentiation. We further delineated the signaling pathways involved and showed that TGF-β1-induced ATF4 production depended on cooperation between canonical TGF-β1 signaling through Smad3 and activation of mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). ATF4, in turn, promoted the transcription of genes encoding enzymes of the de novo serine-glycine biosynthetic pathway and glucose transporter 1 (GLUT1). Our findings suggest that targeting the TGF-β1-mTORC1-ATF4 axis may represent a novel therapeutic strategy for interfering with myofibroblast function in fibrosis and potentially in other conditions, including cancer.

Generation and characterization of a myeloid cell‐specific Cyp19a1 knockout mouse model

CYP19A1 (the aromatase) is a key enzyme in the biosynthesis of estrogens. Estrogens have important regulatory functions, including roles as a major mitogenic stimulus for cellular proliferation. It has been proposed that macrophage CYP19A1 can increase estrogen production at sites of tissue injury, thereby influencing the balance between normal tissue repair and pathogenic outcomes such as hyperproliferation and oncogenic transformation following xenobiotic‐induced toxicity. Our goal is to test this hypothesis using a cell‐specific Cyp19a1‐null mouse model. Here we report the generation and initial characterization of a myeloid cell (including macrophage) specific Cyp19a1‐null mouse model. A newly generated Cyp19a1‐lox mouse was intercrossed with a Cre‐transgenic mouse for myeloid cell specific deletion of a critical CYP19A1 exon. Cyp19a1 deletion was confirmed by PCR analysis of genomic DNA isolated from macrophages in lung bronchoalveolar lavage fluid; according to results of a quantitative PCR analysis, the deletion efficiency was greater than 90% in these cells. Expression of CYP19A1 protein was detected in alveolar macrophages from wild‐type mice, but not in macrophages from Cyp19a1‐null mice, by immunohistochemical and immunofluorescence staining with an anti‐CYP19A1 antibody. Identity of macrophages was confirmed by staining with an anti‐CD14 (macrophage biomarker) antibody. The ability of macrophages in lung bronchoalveolar lavage fluid from wild‐type mice to synthesize estradiol from testosterone was demonstrated using in vitro assays and LC‐MS based detection of estradiol. The aromatase activity was not detected in macrophages from the null mice. Tissue specificity of Cyp19a1 deletion was demonstrated by the lack of detection of the null allele by a quantitative PCR analysis of genomic DNAs from multiple other sources, including lung, liver, ovary, testis, and tail. Homozygous null mice are fertile and show normal growth and activity. They are currently studied for sensitivity to the lung toxicity of naphthalene, a ubiquitous pollutant and possible human lung carcinogen.Support or Funding InformationSupported in part by NIH grant ES020867This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Imaging Visualizes Recruitment of Inflammatory Monocytes and Macrophages to the Injured Heart.

Paradigm shifting studies have revealed that the heart contains functionally diverse populations of macrophages derived from distinct embryonic and adult hematopoietic progenitors. Under steady-state conditions, the heart is largely populated by CCR2- (C-C chemokine receptor type 2) macrophages of embryonic descent. After tissue injury, a dramatic shift in macrophage composition occurs whereby CCR2+ monocytes are recruited to the heart and differentiate into inflammatory CCR2+ macrophages that contribute to heart failure progression. Currently, there are no techniques to noninvasively detect CCR2+ monocyte recruitment into the heart and thus identify patients who may be candidates for immunomodulatory therapy. To develop a noninvasive molecular imaging strategy with high sensitivity and specificity to visualize inflammatory monocyte and macrophage accumulation in the heart. We synthesized and tested the performance of a positron emission tomography radiotracer (68Ga-DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ECL1i [extracellular loop 1 inverso]) that allosterically binds to CCR2. In naive mice, the radiotracer was quickly cleared from the blood and displayed minimal retention in major organs. In contrast, biodistribution and positron emission tomography demonstrated strong myocardial tracer uptake in 2 models of cardiac injury (diphtheria toxin induced cardiomyocyte ablation and reperfused myocardial infarction). 68Ga-DOTA-ECL1i signal localized to sites of tissue injury and was independent of blood pool activity as assessed by quantitative positron emission tomography and ex vivo autoradiography. 68Ga-DOTA-ECL1i uptake was associated with CCR2+ monocyte and CCR2+ macrophage infiltration into the heart and was abrogated in CCR2-/- mice, demonstrating target specificity. Autoradiography demonstrated that 68Ga-DOTA-ECL1i specifically binds human heart failure specimens and with signal intensity associated with CCR2+ macrophage abundance. These findings demonstrate the sensitivity and specificity of 68Ga-DOTA-ECL1i in the mouse heart and highlight the translational potential of this agent to noninvasively visualize CCR2+ monocyte recruitment and inflammatory macrophage accumulation in patients.

Ion Channel Pharmacology for Pain Modulation.

A large series of different ion channels have been identified and investigated as potential targets for new medicines for the treatment of a variety of human diseases, including pain. Among these channels, the voltage gated calcium channels (VGCC) are inhibited by drugs for the treatment of migraine, neuropathic pain or intractable pain. Transient receptor potential (TRP) channels are emerging as important pain transducers as they sense low pH media or oxidative stress and other mediators and are abundantly found at sites of inflammation or tissue injury. Low pH may also activate acid sensing ion channels (ASIC) and mechanical forces stimulate the PIEZO channels. While potent agonists of TRP channels due to their desensitizing action on pain transmission are used as topical applications, the potential of TRP antagonists as pain therapeutics remains an exciting field of investigation. The study of ASIC or PIEZO channels in pain signaling is in an early stage, whereas antagonism of the purinergic P2X3 channels has been reported to provide beneficial effects in chronic intractable cough. The present chapter covers these intriguing channels in great detail, highlighting their diverse mechanisms and broad potential for therapeutic utility.

Role of macrophages in peripheral nerve injury and repair.

Resident and inflammatory macrophages are essential effectors of the innate immune system. These cells provide innate immune defenses and regulate tissue and organ homeostasis. In addition to their roles in diseases such as cancer, obesity and osteoarthritis, they play vital roles in tissue repair and disease rehabilitation. Macrophages and other inflammatory cells are recruited to tissue injury sites where they promote changes in the microenvironment. Among the inflammatory cell types, only macrophages have both pro-inflammatory (M1) and anti-inflammatory (M2) actions, and M2 macrophages have four subtypes. The co-action of M1 and M2 subtypes can create a favorable microenvironment, releasing cytokines for damaged tissue repair. In this review, we discuss the activation of macrophages and their roles in severe peripheral nerve injury. We also describe the therapeutic potential of macrophages in nerve tissue engineering treatment and highlight approaches for enhancing M2 cell-mediated nerve repair and regeneration.

Role of Macrophages in Acute Lung Injury and Chronic Fibrosis Induced by Pulmonary Toxicants.

A diverse group of toxicants has been identified that cause injury to the lung including gases (eg, ozone, chlorine), particulates/aerosols (eg, diesel exhaust, fly ash, other combustion products, mustards, nanomaterials, silica, asbestos), chemotherapeutics (eg, bleomycin), and radiation. The pathologic response to these toxicants depends on the dose and duration of exposure and their physical/chemical properties. A common response to pulmonary toxicant exposure is an accumulation of proinflammatory/cytotoxic M1 macrophages at sites of tissue injury, followed by the appearance of anti-inflammatory/wound repair M2 macrophages. It is thought that the outcome of the pathogenic responses to toxicants depends on the balance in the activity of these macrophage subpopulations. Overactivation of either M1 or M2 macrophages leads to injury and disease pathogenesis. Thus, the very same macrophage-derived mediators, released in controlled amounts to destroy injurious materials and pathogens (eg, reactive oxygen species, reactive nitrogen species, proteases, tumor necrosis factor α) and initiate wound repair (eg, transforming growth factor β, connective tissue growth factor, vascular endothelial growth factor), can exacerbate acute lung injury and/or induce chronic disease such as fibrosis, chronic obstructive pulmonary disease, and asthma, when released in excess. This review focuses on the role of macrophage subsets in acute lung injury and chronic fibrosis. Understanding how these pathologies develop following exposure to toxicants, and the contribution of resident and inflammatory macrophages to disease pathogenesis may lead to the development of novel approaches for treating lung diseases.

TAFA2 Induces Skeletal (Stromal) Stem Cell Migration Through Activation of Rac1-p38 Signaling.

Understanding the mechanisms regulating recruitment of human skeletal (stromal or mesenchymal) stem cells (hMSC) to sites of tissue injury is a prerequisite for their successful use in cell replacement therapy. Chemokine‐like protein TAFA2 is a recently discovered neurokine involved in neuronal cell migration and neurite outgrowth. Here, we demonstrate a possible role for TAFA2 in regulating recruitment of hMSC to bone fracture sites. TAFA2 increased the in vitro trans‐well migration and motility of hMSC in a dose‐dependent fashion and induced significant morphological changes including formation of lamellipodia as revealed by high‐content‐image analysis at single‐cell level. Mechanistic studies revealed that TAFA2 enhanced hMSC migration through activation of the Rac1‐p38 pathway. In addition, TAFA2 enhanced hMSC proliferation, whereas differentiation of hMSC toward osteoblast and adipocyte lineages was not altered. in vivo studies demonstrated transient upregulation of TAFA2 gene expression during the inflammatory phase of fracture healing in a closed femoral fracture model in mice, and a similar pattern was observed in serum levels of TAFA2 in patients after hip fracture. Finally, interleukin‐1β was found as an upstream regulator of TAFA2 expression. Our findings demonstrate that TAFA2 enhances hMSC migration and recruitment and thus is relevant for regenerative medicine applications. Stem Cells 2019;37:407–416

Bone Marrow Fibrocytes Overexpress Gli1 Oncogenic Transcription Factor in Primary Myelofibrosis

Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by myeloid cell proliferation and progressive bone marrow (BM) fibrosis. The degree of the BM fibrosis correlates with unfavorable clinical characteristics and short survival. Whereas it was thought that BM fibrosis in PMF is secondary, caused by mesenchymal stromal cells (MSC) stimulated by cytokine-producing neoplastic megakaryocytes and platelets, we have recently found that BM fibrosis in PMF is induced by neoplastic monocyte-derived fibrocytes which, like MSC, produce collagen-I, collagen-III and fibronectin. Using a PMF immunodeficient mouse model, we demonstrated that inhibition of monocyte differentiation into fibrocytes ameliorates BM fibrosis. Although an increased number of fibrocytes was detected in the BM of patients with PMF, the mechanisms that induce collagen overproduction are still unknown. Glioma oncogene homolog 1 (Gli1), a transcription factor of the hedgehog signaling pathway, activates genes affecting cell migration and tissue fibrosis. Gene expression analyses identified matrix metalloprotease 9 (MMP9) as a key effector of Gli1 that promotes fibrocyte migration into sites of tissue injury. A recent study demonstrated that knockout or inhibition of Gli1 alleviates BM fibrosis. Since it was thought that BM fibrosis is induced by MSC, the function of other BM-derived Gli1-expressingcells was not investigated. Because fibrocytes induce BM fibrosis in PMF, we sought to evaluate the levels of Gli1 in PMF BM cells and in BM-derived fibrocytes and MSC.Methods: BM trephine biopsies and aspirates were obtained from randomly selected PMF patients and healthy controls. Opal multiplex fluorescence IHC was performed on tissue sections using fibrocyte (CD45, CD68, procollagen type I)- and MSC (CD90, CD105)-specific monoclonal antibodies in combination with validated anti-Gli1 antibodies. Single BM cells were classified based on image pattern analysis and their Gli1 signal intensity was assessed. In addition, Low-density cells from BM aspirates were cultured using fibrocyte and MSC culture assays. Following confirmatory immunostaining analysis, RNA was extracted and analyzed using quantitative RT-PCR.Results: Gli1 intensity in fibrocytes of PMF BM biopsies was significantly higher than Gli1 intensity in fibrocytes of healthy control BM biopsies (median: 11.11 vs. 7.16 counts per cell, P=0.02). The median Gli1 signal intensity/cell was 4.3 counts higher in PMF fibrocytes than in PMF MSC (95%CI: 0.5-11.75; P=0.033), whereas Gli1 intensity/cell of PMF MSC was similar to that of normal BM MSC. Similarly, Gli1 mRNA levels were significantly higher in cultured PMF BM-derived fibrocytes than in cultured normal BM-derived fibrocytes (7.16±1.35 vs. 1±0.55, P=0.007). In addition, mRNA levels were higher in PMF BM-derived fibrocytes than in PMF BM-derived MSC (7.16±1.35 vs. 1.36±0.23, P=0.006). Downstream activity of Gli1 in cultured cells was assessed by quantitating the expression of the MMP9 gene. Remarkably, PMF BM-derived fibrocytes expressed significantly higher MMP9 mRNA levels compared to normal BM-derived fibrocytes (12.47±2.25 vs. 1±0.45, P=0.003). In contrast, in PMF BM-derived MSC MMP9 mRNA levels were only 0.03±0.01, 12-fold lower than those of PMF BM-derived fibrocytes (P<0.001) and similar to MMP9 mRNA levels in normal BM-derived MSC.Conclusions: Because Gli1 protein is invariably overrepresented in fibrocytes but not in MSC and Gli1, while MMP9 mRNA levels are higher in PMF BM-derived fibrocytes than in MSC, and considering previous Gli1 gene knockout studies, it is reasonable to assume that Gli1+ BM fibrocytes rather than MSC are the major contributors to the induction of BM fibrosis in PMF. DisclosuresVerstovsek:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

9 - Chlorination and oxidation of human plasma fibronectin by myeloperoxidase-derived oxidants, and its consequences for smooth muscle cell function

Background Fibronectin (FN) occurs as both a soluble form, in plasma and at sites of tissue injury, and a cellular form in tissue extracellular matrices (ECM). FN is critical to wound repair, ECM structure and assembly, cell adhesion and proliferation. FN is reported to play a critical role in the development, progression and stability of cardiovascular atherosclerotic lesions, with high FN levels associated with a thick fibrotic cap, stable disease and a low risk of rupture. Evidence has been presented for FN modification by inflammatory oxidants, and particularly myeloperoxidase (MPO)-derived species including hypochlorous acid (HOCl). The targets and consequences of FN modification are poorly understood. Hypothesis That HOCl and MPO-derived oxidants generate specific modifications (oxidation and chlorination) on FN, and that this alters the adhesion, proliferation and gene expression in human coronary artery smooth muscle cells. Results Mass spectrometry using a novel peptide mass mapping protocol show that HOCl and an enzymatic MPO system, generate site-specific, dose-dependent, chlorination and dichlorination of Tyr residues (up to 16 of 100 residues modified), and oxidation of Trp (7 of 39 residues), Met (3 of 26) and His (1 of 55) residues. This damage occurs within selected FN domains, and particularly the heparin- and cell-binding regions. These alterations increase FN binding to heparin-containing columns. Studies using primary human coronary artery smooth muscle cells (HCASMC) show that exposure to HOCl-modified FN, results in decreased adherence, increased proliferation and altered expression of genes involved in ECM synthesis and remodelling. Conclusions These findings indicate that the presence of modified fibronectin may play a major role in the formation, development and stabilization of fibrous caps in atherosclerotic lesions and may play a key role in the switching of quiescent contractile smooth muscle cells to a migratory, synthetic and proliferative phenotype.

An Efficient Modelling-Simulation-Analysis Workflow to Investigate Stump-Socket Interaction Using Patient-Specific, Three-Dimensional, Continuum-Mechanical, Finite Element Residual Limb Models.

The lack of an efficient modelling-simulation-analysis workflow for creating and utilising detailed subject-specific computational models is one of the key reasons why simulation-based approaches for analysing socket-stump interaction have not yet been successfully established. Herein, we propose a novel and efficient modelling-simulation-analysis workflow that uses commercial software for generating a detailed subject-specific, three-dimensional finite element model of an entire residual limb from Diffusion Tensor MRI images in <20 min. Moreover, to complete the modelling-simulation-analysis workflow, the generated subject-specific residual limb model is used within an implicit dynamic FE simulation of bipedal stance to predict the potential sites of deep tissue injury. For this purpose, a nonlinear hyperelastic, transversely isotropic skeletal muscle constitutive law containing a deep tissue injury model was implemented in LS-DYNA. To demonstrate the feasibility of the entire modelling-simulation-analysis workflow and the fact that detailed, anatomically realistic, multi-muscle models are superior to state-of-the-art, fused-muscle models, an implicit dynamic FE analysis of 2-h bipedal stance is carried out. By analysing the potential volume of damaged muscle tissue after donning an optimally-fitted and a misfitted socket, i.e., a socket whose volume was isotropically shrunk by 10%, we were able to highlight the differences between the detailed individual- and fused-muscle models. The results of the bipedal stance simulation showed that peak stresses in the fused-muscle model were four times lower when compared to the multi-muscle model. The peak interface stress in the individual-muscle model, at the end of bipedal stance analysis, was 2.63 times lower than that in the deep tissues of the stump. At the end of the bipedal stance analysis using the misfitted socket, the fused-muscle model predicted that 7.65% of the residual limb volume was injured, while the detailed-model predicted 16.03%. The proposed approach is not only limited to modelling residual limbs but also has applications in predicting the impact of plastic surgery, for detailed forward-dynamics simulations of normal musculoskeletal systems.

Effects of mesenchymal stromal cells on type 1 diabetes mellitus rat muscles.

Type 1 diabetes mellitus (DM) causes marked skeletal muscle atrophy. Mesenchymal stromal cells (MSC) are an attractive therapy to avoid diabetic complications because of their ability to modify the microenvironment at sites of tissue injury. The objective of this study was to evaluate the effects of MSC transplantation on muscle adaptation caused by diabetes. DM was induced by streptozotocin (STZ), and the diabetic animals received systemic MSC transplantation. The von Frey test and footprint analysis were used to assess sensation and sensory motor performance, respectively. Tibialis anterior muscles were investigated by morphology; molecular markers atrogin-1/muscle RING-finger protein-1, nuclear factor κB/p38 mitogen-activated protein kinase, tumor necrosis-like weak inducer of apoptosis/fibroblast growth factor-inducible 14, myostatin, myogenic differentiation 1, and insulin-like growth factor 1 were also assessed. MSC transplantation improved sensation and walking performance and also decreased muscle fibrosis in DM rats by modulating atrogenes but did not prevent muscle atrophy. MSCs can reduce muscle and functional complications that result from type 1 DM in rats. Muscle Nerve 58: 583-591, 2018.

Induction of antiinflammatory purinergic signaling in activated human iNKT cells.

Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define "purinergic Th2-type cytokine bias" as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.

Chlorination and oxidation of human plasma fibronectin by myeloperoxidase-derived oxidants, and its consequences for smooth muscle cell function

Fibronectin (FN) occurs as both a soluble form, in plasma and at sites of tissue injury, and a cellular form in tissue extracellular matrices (ECM). FN is critical to wound repair, ECM structure and assembly, cell adhesion and proliferation. FN is reported to play a critical role in the development, progression and stability of cardiovascular atherosclerotic lesions, with high FN levels associated with a thick fibrotic cap, stable disease and a low risk of rupture. Evidence has been presented for FN modification by inflammatory oxidants, and particularly myeloperoxidase (MPO)-derived species including hypochlorous acid (HOCl). The targets and consequences of FN modification are poorly understood. Here we show, using a newly-developed MS protocol, that HOCl and an enzymatic MPO system, generate site-specific dose-dependent Tyr chlorination and dichlorination (up to 16 of 100 residues modified), and oxidation of Trp (7 of 39 residues), Met (3 of 26) and His (1 of 55) within selected FN domains, and particularly the heparin- and cell-binding regions. These alterations increase FN binding to heparin-containing columns. Studies using primary human coronary artery smooth muscle cells (HCASMC) show that exposure to HOCl-modified FN, results in decreased adherence, increased proliferation and altered expression of genes involved in ECM synthesis and remodelling. These findings indicate that the presence of modified fibronectin may play a major role in the formation, development and stabilisation of fibrous caps in atherosclerotic lesions and may play a key role in the switching of quiescent contractile smooth muscle cells to a migratory, synthetic and proliferative phenotype.

PGE2 production at sites of tissue injury promotes an anti-inflammatory neutrophil phenotype and determines the outcome of inflammation resolution in vivo.

Neutrophils are the first immune cells recruited to a site of injury or infection, where they perform many functions. Having completed their role, neutrophils must be removed from the inflammatory site-either by apoptosis and efferocytosis or by reverse migration away from the wound-for restoration of normal tissue homeostasis. Disruption of these tightly controlled physiological processes of neutrophil removal can lead to a range of inflammatory diseases. We used an in vivo zebrafish model to understand the role of lipid mediator production in neutrophil removal. Following tailfin amputation in the absence of macrophages, neutrophillic inflammation does not resolve, due to loss of macrophage-dependent handling of eicosanoid prostaglandin E2 (PGE2) that drives neutrophil removal via promotion of reverse migration. Knockdown of endogenous PGE synthase gene reveals PGE2 as essential for neutrophil inflammation resolution. Furthermore, PGE2 is able to signal through EP4 receptors during injury, causing an increase in Alox12 production and switching toward anti-inflammatory eicosanoid signaling. Our data confirm regulation of neutrophil migration by PGE2 and LXA4 (lipoxin A4) in an in vivo model of inflammation resolution. This pathway may contain therapeutic targets for driving inflammation resolution in chronic inflammatory disease.

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications

BackgroundThe regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. MethodsBM-hMSCs were expanded using xenogenic-free media and exofucosylated using α(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. ResultsOur protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. DiscussionThe described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.

Silk Fibroin Porous Scaffolds Loaded with a Slow-Releasing Hydrogen Sulfide Agent (GYY4137) for Applications of Tissue Engineering.

Hydrogen sulfide (H2S) is a physiological gasotransmitter known to possess a regulatory role in several tissues, including bone. The exogenous administration by injection of solutions of H2S-releasing compounds (e.g., GYY4137) has been previously investigated as a novel therapeutic approach for the treatment of bone diseases. Here, GYY4137 was embedded into fibroin sponges, previously shown to be suitable as scaffolds for bone, thanks to their biocompatibility, scalable porous structure, and biodegradability rate. Fibroin porous scaffolds were produced by solvent casting and the particulate leaching method, and GYY4137 was successively incorporated by using dimethyl sulfoxide (DMSO) as vehicle. The process used to produce GYY4137-loaded scaffolds allowed the incorporation of different controlled amounts of GYY4137 into fibroin matrices. The loading process preserved the properties of the system components in the final products, as assessed by SEM, FT-IR, NMR, and different thermal analyses techniques. Release of H2S from GYY4137 incorporated into the scaffolds was monitored upon incubation in saline solution at physiological pH: H2S-release kinetic was found to be dependent on the amount of GYY4137. To ensure biocompatibility, mouse fibroblasts and human primary bone marrow stromal cells were seeded onto scaffolds, and short-term viability assays were performed. Results showed that the GYY4137-loaded scaffold did not induce cytotoxicity in any of the cell type tested. Our findings demonstrate that embedding an H2S-releasing donor in silk fibroin scaffold is a suitable strategy to achieve a long-lasting release of H2S that preserves cell viability and allows local delivery at sites of tissue injury.

Ergothioneine - a diet-derived antioxidant with therapeutic potential.

Ergothioneine is a thiol/thione molecule synthesised only by some fungi and bacteria. Nonetheless, it is avidly taken up from the diet by humans and other animals through a transporter, OCTN1, and accumulates to high levels in certain tissues. Ergothioneine is not rapidly metabolised, or excreted in urine and is present in many, if not all, human tissues and body fluids. Ergothioneine has powerful antioxidant and cytoprotective properties in vitro and there is evidence that the body may concentrate it at sites of tissue injury by raising OCTN1 levels. Decreased blood and/or plasma levels of ergothioneine have been observed in some diseases, suggesting that a deficiency could be relevant to the disease onset or progression. This brief Review explores the possible roles of ergothioneine in human health and disease.

The role of the epithelial-to-mesenchymal transition (EMT) in diseases of the salivary glands.

The link between inflammatory microenvironment and cancer emerged in the last years as a decisive factor in the induction of the pathological epithelial-mesenchymal transition (EMT). The EMT induces changes of cell states converting the epithelial cells to mesenchymal cells when this program is fully executed and EMT has emerged as a central driver of tumor malignancy. Cellular pathways activated by chronic inflammation brought about by chronic infections, by immune-mediated diseases, or by dysregulated wound healing at sites of repetitive tissue injury, constitute risk factors or initial cell transformation and for cancer progression. EMT and its intermediate states have recently been identified as crucial inducers of organ fibrosis, inflammation and tumor progression. In this review, we discuss the current state-of-the-art and latest findings regarding the link between EMT, inflammation, fibrosis and cancer, highlighting the most recent data on EMT-dependent tissue fibrosis during chronic inflammatory salivary glands conditions and salivary glands tumors.

Innate effector cells in angiogenesis and lymphangiogenesis.

Angiogenesis and lymphangiogenesis are distinct and complex processes requiring a finely tuned balance between stimulatory and inhibitory signals. During adulthood, angiogenesis and lymphangiogenesis are activated at sites of tumor growth, tissue injury and remodeling, and chronic inflammation. Vascular endothelial growth factors (VEGFs), angiopoietin (ANGPTs) and a multitude of additional signaling molecules play distinct roles in the modulation of angiogenesis/lymphangiogenesis. VEGFs and ANGPTs activate specific tyrosine kinase receptor (e.g., VEGFR1, VEGFR-2, VEGFR-3 and TIE2 respectively), expressed on blood endothelial cells (angiogenesis) and lymphatic endothelial cells (lymphangiogenesis). Although tumor cells produce VEGFs and other proangiogenic mediators, tissue resident (e.g., macrophages, mast cells) and circulating immune cells (e.g., basophils, neutrophils, monocytes, eosinophils) are an important source of angiogenic/lymphangiogenic mediators in inflammation and in tumor microenvironment and at site of chronic inflammation. Certain immune cells can also release anti-angiogenic factors. Mast cells, basophils, neutrophils and presumably other immune cells are not only a source of angiogenic/lymphangiogenic molecules, but also their target. Cells of the immune system need consideration as major players and possible targets for therapeutic manipulation of angiogenesis/lymphangiogenesis in chronic inflammatory disorders and tumors.