Thomas Bertalot

Anti-inflammatory activity of Wnt signaling in enteric nervous system: in vitro preliminary evidences in rat primary cultures.

BackgroundIn the last years, Wnt signaling was demonstrated to regulate inflammatory processes. In particular, an increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor κB (NF-кB), TNFa release, and IL10 expression.MethodsTo investigate the role of Wnt pathway in the response of the enteric nervous system (ENS) to inflammation, neurons and glial cells from rat myenteric plexus were treated with exogenous Wnt3a and/or LPS with or without supporting neurotrophic factors such as basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). The immunophenotypical characterization by flow cytometry and the protein and gene expression analysis by qPCR and Western blotting were carried out.ResultsFlow cytometry and immunofluorescence staining evidenced that enteric neurons coexpressed Frizzled 9 and toll-like receptor 4 (TLR4) while glial cells were immunoreactive to TLR4 and Wnt3a suggesting that canonical Wnt signaling is active in ENS.Under in vitro LPS treatment, Western blot analysis demonstrated an active cross talk between canonical Wnt signaling and NF-кB pathway that is essential to negatively control enteric neuronal response to inflammatory stimuli. Upon costimulation with LPS and Wnt3a, a significant anti-inflammatory activity was detected by RT-PCR based on an increased IL10 expression and a downregulation of pro-inflammatory cytokines TNFa, IL1B, and interleukin 6 (IL6). When the availability of neurotrophic factors in ENS cultures was abolished, a changed cell reactivity by Wnt signaling was observed at basal conditions and after LPS treatment.ConclusionsThe results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. Moreover, experimental evidences were provided to clarify the correlation among soluble trophic factors, Wnt signaling, and anti-inflammatory protection of ENS.

Systematic Review of Pharmacological Properties of the Oligodendrocyte Lineage

Oligodendrogenesis and oligodendrocyte precursor maturation are essential processes during the course of central nervous system development, and lead to the myelination of axons. Cells of the oligodendrocyte lineage are generated in the germinal zone from migratory bipolar oligodendrocyte precursor cells (OPCs), and acquire cell surface markers as they mature and respond specifically to factors which regulate proliferation, migration, differentiation, and survival. Loss of myelin underlies a wide range of neurological disorders, some of an autoimmune nature—multiple sclerosis probably being the most prominent. Current therapies are based on the use of immunomodulatory agents which are likely to promote myelin repair (remyelination) indirectly by subverting the inflammatory response, aspects of which impair the differentiation of OPCs. Cells of the oligodendrocyte lineage express and are capable of responding to a diverse array of ligand-receptor pairs, including neurotransmitters and nuclear receptors such as γ-aminobutyric acid, glutamate, adenosine triphosphate, serotonin, acetylcholine, nitric oxide, opioids, prostaglandins, prolactin, and cannabinoids. The intent of this review is to provide the reader with a synopsis of our present state of knowledge concerning the pharmacological properties of the oligodendrocyte lineage, with particular attention to these receptor-ligand (i.e., neurotransmitters and nuclear receptor) interactions that can influence oligodendrocyte migration, proliferation, differentiation, and myelination, and an appraisal of their therapeutic potential. For example, many promising mediators work through Ca2+ signaling, and the balance between Ca2+ influx and efflux can determine the temporal and spatial properties of oligodendrocytes (OLs). Moreover, Ca2+ signaling in OPCs can influence not only differentiation and myelination, but also process extension and migration, as well as cell death in mature mouse OLs. There is also evidence that oligodendroglia exhibit Ca2+ transients in response to electrical activity of axons for activity-dependent myelination. Cholinergic antagonists, as well as endocannabinoid-related lipid-signaling molecules target OLs. An understanding of such pharmacological pathways may thus lay the foundation to allow its leverage for therapeutic benefit in diseases of demyelination.

Histone deacetylase inhibitors restore IL-10 expression in lipopolysaccharide-induced cell inflammation and reduce IL-1β and IL-6 production in breast silicone implant in C57BL/6J wild-type murine model.

Among epigenetic enzymes, histone deacetylases (HDACs) are responsible for regulating the expression of an extensive array of genes by reversible deacetylation of nuclear histones as well as a large number of non-histone proteins. Initially proposed for cancer therapy, recently the interest for HDAC inhibitors (HDACi) as orally active, safe, and anti-inflammatory agents is rising due to their ability in reducing the severity of inflammatory and autoimmune diseases. In particular, selective HDAC3, HDAC6, and HDAC8 inhibitors have been described to downregulate the expression of pro-inflammatory cytokines (TNF-α, TGF-β, IL-1β, and IL-6). Herein, using KB31, C2C12, and 3T3-J2 cell lines, we demonstrated that, under lipopolysaccharide-induced in vitro inflammation, HDAC3/6/8 inhibitor MC2625 and HDAC6-selective inhibitor MC2780 were effective at a concentration of 30 ng/mL to downregulate mRNA expression of pro-inflammatory cytokines (IL-1β and IL-6) and to promote the transcription of IL-10 gene, without affecting the cell viability. Afterwards, we investigated by immunohistochemistry the activity of MC2625 and MC2780 at a concentration of 60 ng/kg animal weight to regulate silicone-triggered immune response in C57BL/6J female mice. Our findings evidenced the ability of such inhibitors to reduce host inflammation in silicone implants promoting a thickness reduction of peri-implant fibrous capsule, upregulating IL-10 expression, and reducing the production of both IL-1β and IL-6. These results underline the potential application of MC2625 and MC2780 in inflammation-related diseases.

Maintenance of the enteric stem cell niche by bacterial lipopolysaccharides? Evidence and perspectives

The enteric nervous system (ENS) has to respond to continuously changing microenvironmental challenges within the gut and is therefore dependent on a neural stem cell niche to keep the ENS functional throughout life. In this study, we hypothesize that this stem cell niche is also affected during inflammation and therefore investigated lipopolysaccharides (LPS) effects on enteric neural stem/progenitor cells (NSPCs). NSPCs were derived from the ENS and cultured under the influence of different LPS concentrations. LPS effects upon proliferation and differentiation of enteric NSPC cultures were assessed using immunochemistry, flow cytometry, western blot, Multiplex ELISA and real‐time PCR. LPS enhances the proliferation of enteric NSPCs in a dose‐dependent manner. It delays and modifies the differentiation of these cells. The expression of the LPS receptor toll‐like receptor 4 on NSPCs could be demonstrated. Moreover, LPS induces the secretion of several cytokines. Flow cytometry data gives evidence for individual subgroups within the NSPC population. ENS‐derived NSPCs respond to LPS in maintaining at least partially their stem cell character. In the case of inflammatory disease or trauma where the liberation and exposure to LPS will be increased, the expansion of NSPCs could be a first step towards regeneration of the ENS. The reduced and altered differentiation, as well as the induction of cytokine signalling, demonstrates that the stem cell niche may take part in the LPS‐transmitted inflammatory processes in a direct and defined way.

Blood vessel-derived acellular matrix for vascular graft application.

To overcome the issues connected to the use of autologous vascular grafts and artificial materials for reconstruction of small diameter (<6 mm) blood vessels, this study aimed to develop acellular matrix- (AM-) based vascular grafts. Rat iliac arteries were decellularized by a detergent-enzymatic treatment, whereas endothelial cells (ECs) were obtained through enzymatic digestion of rat skin followed by immunomagnetic separation of CD31-positive cells. Sixteen female Lewis rats (8 weeks old) received only AM or previously in vitro reendothelialized AM as abdominal aorta interposition grafts (about 1 cm). The detergent-enzymatic treatment completely removed the cellular part of vessels and both MHC class I and class II antigens. One month after surgery, the luminal surface of implanted AMs was partially covered by ECs and several platelets adhered in the areas lacking cell coverage. Intimal hyperplasia, already detected after 1 month, increased at 3 months. On the contrary, all grafts composed by AM and ECs were completely covered at 1 month and their structure was similar to that of native vessels at 3 months. Taken together, our findings show that prostheses composed of AM preseeded with ECs could be a promising approach for the replacement of blood vessels.

Neuronal commitment of human circulating multipotent cells by carbon nanotube-polymer scaffolds and biomimetic peptides

AIM We aimed to set up a self-standing, biomimetic scaffold system able to induce and support per se neuronal differentiation of autologous multipotent cells. MATERIALS & METHODS We isolated a population of human circulating multipotent cells (hCMCs), and used carbon nanotube/polymer nanocomposite scaffolds to mimic electrical/nanotopographical features of the neural environment, and biomimetic peptides reproducing axon guidance cues from neural proteins. RESULTS hCMCs showed high degree of stemness and multidifferentiative potential; stimuli from the scaffolds and biomimetic peptides could induce and boost hCMC differentiation toward neuronal lineage despite the absence of exogenously added, specific growth factors. CONCLUSION This work suggests the scaffold-peptides system combined with autologous hCMCs as a functional biomimetic, self-standing prototype for neural regenerative medicine applications.

Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells.

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality.

Leucocyte and Platelet-rich Fibrin: a carrier of autologous multipotent cells for regenerative medicine

The wound healing is a complex process wherein inflammation, proliferation and regeneration evolve according to a spatio‐temporal pattern from the activation of coagulation cascade to the formation of a plug clot including fibrin matrix, blood‐borne cells and cytokines/growth factors. Creating environments conducive to tissue repair, the haemoderivatives are commonly proposed for the treatment of hard‐to‐heal wounds. Here, we explored in vitro the intrinsic regenerative potentialities of a leucocyte‐ and platelet‐rich fibrin product, known as CPL‐MB, defining the stemness grade of cells sprouting from the haemoderivative. Using highly concentrated serum‐based medium to simulate wound conditions, we isolated fibroblast‐like cells (CPL‐CMCs) adhering to plastic and showing stable in vitro propagation, heterogeneous stem cell expression pattern, endothelial adhesive properties and immunomodulatory profile. Due to their blood derivation and expression of CXCR4, CPL‐CMCs have been suggested to be immature cells circulating in peripheral blood at quiescent state until activation by both coagulation event and inflammatory stimuli such as stromal‐derived factor 1/SDF1. Expressing integrins (CD49f, CD103), vascular adhesion molecules (CD106, CD166), endoglin (CD105) and remodelling matrix enzymes (MMP2, MMP9, MMP13), they showed a transendothelial migratory potential besides multipotency. Taken together, our data suggested that a standardized, reliable and economically feasible blood product such as CPL‐MB functions as an artificial stem cell niche that, under permissive conditions, originate ex vivo immature cells that could be useful for autologous stem cell‐based therapies.

Adrenomedullin in the growth modulation and differentiation of acute myeloid leukemia cells

Adrenomedullin (ADM) is a regulatory peptide endowed with multiple biological effects, including the regulation of blood pressure, cell growth and innate host defence. In the present study, we demonstrated that ADM signaling could be involved in the impaired cellular differentiation of myeloid leukemia cells to mature granulocytes or monocytes by modulating RAMPs/CRLR expression, PI3K/Akt cascade and the ERK/MAPK signaling pathway. When exogenously administered to in vitro cultures of HL60 promyelocytic leukemia cells, ADM was shown to exert a strong proliferative effect with minimal upregulation in the expression level of monocyte antigen CD14. Notably, the experimental inhibition of ADM signaling with inhibitor ADM22-52 promoted a differentiative stimulation towards monocytic and granulocytic lineages. Moreover, based on the expression of CD31 relative to CD38, we hypothesized that an excess of ADM in bone marrow (BM) niche could increase the transendothelial migration of leukemia cells while any inhibitory event of ADM activity could raise cell retention in hyaluronate matrix by upregulating CD38. Taken into consideration the above evidence, we concluded that ADM and ADM22-52 could differently affect the growth of leukemia cells by autocrine/paracrine mechanisms and may have clinical relevance as biological targets for the intervention of tumor progression.