Publications by authors named "Philippe Naveilhan"

39 Publications

T cells show preferential adhesion to enteric neural cells in culture and are close to neural cells in the myenteric ganglia of Crohn's patients.

J Neuroimmunol 2020 12 7;349:577422. Epub 2020 Oct 7.

Université de Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France.

Plexitis in the proximal margin of intestinal resections are associated with post-operative recurrence of Crohn's disease. To understand their formation, in vitro analyzes were performed. T cells adhered preferentially to neuron and glial cells in mixed primary cultures of enteric nervous system and T cell activation increased their adhesion capacity. Higher number of T lymphocytes in close proximity to enteric glial cells was also observed in the myenteric ganglia of Crohn's patients as compared to control. These data show that close proximity between lymphocytes and enteric neural cells exists and may contribute to the formation of plexitis.
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http://dx.doi.org/10.1016/j.jneuroim.2020.577422DOI Listing
December 2020

IL-7 receptor influences anti-TNF responsiveness and T cell gut homing in inflammatory bowel disease.

J Clin Invest 2019 04 2;129(5):1910-1925. Epub 2019 Apr 2.

OSE Immunotherapeutics, Nantes, France.

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti-TNF, or anti-α4β7 therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled α4β7 integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment.
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http://dx.doi.org/10.1172/JCI121668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486337PMC
April 2019

Rat enteric glial cells express novel isoforms of Interleukine-7 regulated during inflammation.

Neurogastroenterol Motil 2019 01 21;31(1):e13467. Epub 2018 Sep 21.

Université de Nantes, INSERM, Institut des Maladies de l'Appareil Digestif, The enteric nervous system in gut and brain disorders, Nantes, France.

Background: Neuroimmune interactions are essential to maintain gut homeostasis and prevent intestinal disorders but so far, the impact of enteric glial cells (EGC) on immune cells remains a relatively unexplored area of research. As a dysregulation of critical cytokines such as interleukine-7 (IL-7) was suggested to exacerbate gut chronic inflammation, we investigated whether EGC could be a source of IL-7 in the gastrointestinal tract.

Methods: Expression of IL-7 in the rat enteric nervous system was analyzed by immunochemistry and Q-PCR. IL-7 variants were cloned and specific antibodies against rat IL-7 isoforms were raised to characterize their expression in the submucosal plexus. IL-7 isoforms were produced in vitro to analyze their impact on T-cell survival.

Key Results: Neurons and glial cells of the rat enteric nervous system expressed IL-7 at both mRNA and protein levels. Novel rat IL-7 isoforms with distinct C-terminal parts were detected. Three of these isoforms were found in EGC or in both enteric neurons and EGC. Exposure of EGC to pro-inflammatory cytokines (IL-1β and/or TNFα) induced an upregulation of all IL-7 isoforms. Interestingly, time-course and intensity of the upregulation varied according to the presence or absence of exon 5a in IL-7 variants. Functional analysis on T lymphocytes revealed that only canonical IL-7 protects T cells from cell death.

Conclusions And Inferences: IL-7 and its variants are expressed by neurons and glial cells in the enteric nervous system. Their distinct expression and upregulation in inflammatory conditions suggest a role in gut homeostasis which could be critical in case of chronic inflammatory diseases.
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http://dx.doi.org/10.1111/nmo.13467DOI Listing
January 2019

The multiple faces of inflammatory enteric glial cells: is Crohn's disease a gliopathy?

Am J Physiol Gastrointest Liver Physiol 2018 07 8;315(1):G1-G11. Epub 2018 Mar 8.

Inserm, UMR1235 TENS, Nantes , France.

Gone are the days when enteric glial cells (EGC) were considered merely satellites of enteric neurons. Like their brain counterpart astrocytes, EGC express an impressive number of receptors for neurotransmitters and intercellular messengers, thereby contributing to neuroprotection and to the regulation of neuronal activity. EGC also produce different soluble factors that regulate neighboring cells, among which are intestinal epithelial cells. A better understanding of EGC response to an inflammatory environment, often referred to as enteric glial reactivity, could help define the physiological role of EGC and the importance of this reactivity in maintaining gut functions. In chronic inflammatory disorders of the gut such as Crohn's disease (CD) and ulcerative colitis, EGC exhibit abnormal phenotypes, and their neighboring cells are dysfunctional; however, it remains unclear whether EGC are only passive bystanders or active players in the pathophysiology of both disorders. The aim of the present study is to review the physiological roles and properties of EGC, their response to inflammation, and their role in the regulation of the intestinal epithelial barrier and to discuss the emerging concept of CD as an enteric gliopathy.
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http://dx.doi.org/10.1152/ajpgi.00016.2018DOI Listing
July 2018

Low-Dose Pesticide Mixture Induces Senescence in Normal Mesenchymal Stem Cells (MSC) and Promotes Tumorigenic Phenotype in Premalignant MSC.

Stem Cells 2017 03 23;35(3):800-811. Epub 2016 Nov 23.

Centre de Recherche en Cancérologie Nantes Angers UMR INSERM 892, CNRS 6299 - Equipe 9, Université de Nantes, Nantes, France.

Humans are chronically exposed to multiple environmental pollutants such as pesticides with no significant evidence about the safety of such poly-exposures. We exposed mesenchymal stem cells (MSC) to very low doses of mixture of seven pesticides frequently detected in food samples for 21 days in vitro. We observed a permanent phenotype modification with a specific induction of an oxidative stress-related senescence. Pesticide mixture also induced a shift in MSC differentiation towards adipogenesis but did not initiate a tumorigenic transformation. In modified MSC in which a premalignant phenotype was induced, the exposure to pesticide mixture promoted tumorigenic phenotype both in vitro and in vivo after cell implantation, in all nude mice. Our results suggest that a common combination of pesticides can induce a premature ageing of adult MSC, and as such could accelerate age-related diseases. Exposure to pesticide mixture may also promote the tumorigenic transformation in a predisposed stromal environment. Abstract Video Link: https://youtu.be/mfSVPTol-Gk Stem Cells 2017;35:800-811.
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http://dx.doi.org/10.1002/stem.2539DOI Listing
March 2017

Enteric glial cells have specific immunosuppressive properties.

J Neuroimmunol 2016 06 18;295-296:79-83. Epub 2016 Apr 18.

INSERM, UMR 913, Nantes F-44035, France; Université de Nantes, Faculté de Médecine, Nantes F-44035, France; CHU de Nantes, Institut des Maladies de l'Appareil Digestif, IMAD, Nantes F-44093, France. Electronic address:

Enteric glial cells (EGC) have trophic and neuroregulatory functions in the enteric nervous system, but whether they exert a direct effect on immune cells is unknown. Here, we used co-cultures to show that human EGC can inhibit the proliferation of activated T lymphocytes. Interestingly, EGC from Crohn's patients were effective at one EGC for two T cells whereas EGC from control patients required a ratio of 1:1. These data suggest that EGC contribute to local immune homeostasis in the gastrointestinal wall. They also raise the possibility that EGC have particular immunosuppressive properties in inflammatory bowel diseases such as Crohn's disease.
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http://dx.doi.org/10.1016/j.jneuroim.2016.04.011DOI Listing
June 2016

Postnatal development of the myenteric glial network and its modulation by butyrate.

Am J Physiol Gastrointest Liver Physiol 2016 06 7;310(11):G941-51. Epub 2016 Apr 7.

INSERM, U913, Nantes, France; Université Nantes, Nantes, France; CHU Nantes, Hôtel Dieu, Institut des Maladies de l'Appareil Digestif, France; and Centre de Recherche en Nutrition Humaine, Nantes, France

The postnatal period is crucial for the development of gastrointestinal (GI) functions. The enteric nervous system is a key regulator of GI functions, and increasing evidences indicate that 1) postnatal maturation of enteric neurons affect the development of GI functions, and 2) microbiota-derived short-chain fatty acids can be involved in this maturation. Although enteric glial cells (EGC) are central regulators of GI functions, the postnatal evolution of their phenotype remains poorly defined. We thus characterized the postnatal evolution of EGC phenotype in the colon of rat pups and studied the effect of short-chain fatty acids on their maturation. We showed an increased expression of the glial markers GFAP and S100β during the first postnatal week. As demonstrated by immunohistochemistry, a structured myenteric glial network was observed at 36 days in the rat colons. Butyrate inhibited EGC proliferation in vivo and in vitro but had no effect on glial marker expression. These results indicate that the EGC myenteric network continues to develop after birth, and luminal factors such as butyrate endogenously produced in the colon may affect this development.
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http://dx.doi.org/10.1152/ajpgi.00232.2015DOI Listing
June 2016

Targeting the CD80/CD86 costimulatory pathway with CTLA4-Ig directs microglia toward a repair phenotype and promotes axonal outgrowth.

Glia 2015 Dec 27;63(12):2298-312. Epub 2015 Jul 27.

INSERM UMR 1064, ITUN, CHU Nantes, University of Nantes, France.

Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia.
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http://dx.doi.org/10.1002/glia.22894DOI Listing
December 2015

Local control of the host immune response performed with mesenchymal stem cells: perspectives for functional intracerebral xenotransplantation.

J Cell Mol Med 2015 Jan 14;19(1):124-34. Epub 2014 Oct 14.

INSERM, UMR 1064, Nantes, France; CHU de Nantes, Institut de Transplantation et de Recherche en Transplantation, ITERT, Nantes, France; Faculté de Médecine, Université de Nantes, LUNAM Université, Nantes, France.

Foetal pig neuroblasts are interesting candidates as a cell source for transplantation, but xenotransplantation in the brain requires the development of adapted immunosuppressive treatments. As systemic administration of high doses of cyclosporine A has side effects and does not protect xenotransplants forever, we focused our work on local control of the host immune responses. We studied the advantage of cotransplanting syngenic mesenchymal stem cells (MSC) with porcine neuroblasts (pNb) in immunocompetent rat striata. Two groups of animals were transplanted, either with pNb alone or with both MSC and pNb. At day 63, no porcine neurons were detected in the striata that received only pNb, while four of six rats transplanted with both pNb and MSC exhibited healthy porcine neurons. Interestingly, 50% of the cotransplanted rats displayed healthy grafts with pNF70+ and TH+ neurons at 120 days post-transplantation. qPCR analyses revealed a general dwindling of pro- and anti-inflammatory cytokines in the striata that received the cotransplants. Motor recovery was also observed following the transplantation of pNb and MSC in a rat model of Parkinson's disease. Taken together, the present data indicate that the immunosuppressive properties of MSC are of great interest for the long-term survival of xenogeneic neurons in the brain.
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http://dx.doi.org/10.1111/jcmm.12414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288356PMC
January 2015

Human dental pulp stem cells cultured in serum-free supplemented medium.

Front Physiol 2013 11;4:357. Epub 2013 Dec 11.

INSERM, UMR1064 ITERT, Institut de Transplantation et de Recherche en Transplantation Nantes, France ; Faculty of Odontology, University of Nantes Nantes, France.

Unlabelled: Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells.

Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR.

Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs.

Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs.
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http://dx.doi.org/10.3389/fphys.2013.00357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858652PMC
December 2013

Survival and differentiation of adenovirus-generated induced pluripotent stem cells transplanted into the rat striatum.

Cell Transplant 2014 22;23(11):1407-23. Epub 2013 Jul 22.

Program in Neuroscience, Field Neurosciences Laboratory for Restorative Neurology Brain Research and Integrative Neuroscience Center, Central Michigan University, Mount Pleasant, MI, USA.

Induced pluripotent stem cells (iPSCs) offer certain advantages over embryonic stem cells in cell replacement therapy for a variety of neurological disorders. However, reliable procedures, whereby transplanted iPSCs can survive and differentiate into functional neurons, without forming tumors, have yet to be devised. Currently, retroviral or lentiviral reprogramming methods are often used to reprogram somatic cells. Although the use of these viruses has proven to be effective, formation of tumors often results following in vivo transplantation, possibly due to the integration of the reprogramming genes. The goal of the current study was to develop a new approach, using an adenovirus for reprogramming cells, characterize the iPSCs in vitro, and test their safety, survivability, and ability to differentiate into region-appropriate neurons following transplantation into the rat brain. To this end, iPSCs were derived from bone marrow-derived mesenchymal stem cells and tail-tip fibroblasts using a single cassette lentivirus or a combination of adenoviruses. The reprogramming efficiency and levels of pluripotency were compared using immunocytochemistry, flow cytometry, and real-time polymerase chain reaction. Our data indicate that adenovirus-generated iPSCs from tail-tip fibroblasts are as efficient as the method we used for lentiviral reprogramming. All generated iPSCs were also capable of differentiating into neuronal-like cells in vitro. To test the in vivo survivability and the ability to differentiate into region-specific neurons in the absence of tumor formation, 400,000 of the iPSCs derived from tail-tip fibroblasts that were transfected with the adenovirus pair were transplanted into the striatum of adult, immune-competent rats. We observed that these iPSCs produced region-specific neuronal phenotypes, in the absence of tumor formation, at 90 days posttransplantation. These results suggest that adenovirus-generated iPSCs may provide a safe and viable means for neuronal replacement therapies.
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http://dx.doi.org/10.3727/096368913X670958DOI Listing
June 2015

Colonic inflammation in Parkinson's disease.

Neurobiol Dis 2013 Feb 24;50:42-8. Epub 2012 Sep 24.

Inserm, U913, Nantes, F-44093, France.

Lewy pathology affects the gastrointestinal tract in Parkinson's disease (PD) and data from recent genetic studies suggest a link between PD and gut inflammation. We therefore undertook the present survey to investigate whether gastrointestinal inflammation occurs in PD patients. Nineteen PD patients and 14 age-matched healthy controls were included. For each PD patients, neurological and gastrointestinal symptoms were assessed using the Unified Parkinson's Disease Rating Scale part III and the Rome III questionnaire, respectively and cumulative lifetime dose of L-dopa was calculated. Four biopsies were taken from the ascending colon during the course of a total colonoscopy in controls and PD patients. The mRNA expression levels of pro-inflammatory cytokines (tumor necrosis factor alpha, interferon gamma, interleukin-6 and interleukin-1 beta) and glial marker (Glial fibrillary acidic protein, Sox-10 and S100-beta) were analyzed using real-time PCR in two-pooled biopsies. Immunohistochemical analysis was performed on the two remaining biopsies using antibodies against phosphorylated alpha-synuclein to detect Lewy pathology. The mRNA expression levels of pro-inflammatory cytokines as well as of two glial markers (Glial fibrillary acidic protein and Sox-10) were significantly elevated in the ascending colon of PD patients with respect to controls. The levels of tumor necrosis factor alpha, interferon gamma, interleukin-6, interleukin-1 beta and Sox-10 were negatively correlated with disease duration. By contrast, no correlations were found between the levels of pro-inflammatory cytokines or glial markers and disease severity, gastrointestinal symptoms or cumulative lifetime dose of L-dopa. There was no significant difference in the expression of pro-inflammatory cytokines or glial marker between patients with and without enteric Lewy pathology. Our findings provide evidence that enteric inflammation occurs in PD and further reinforce the role of peripheral inflammation in the initiation and/or the progression of the disease.
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http://dx.doi.org/10.1016/j.nbd.2012.09.007DOI Listing
February 2013

Expression of heme oxygenase-1 in neural stem/progenitor cells as a potential mechanism to evade host immune response.

Stem Cells 2012 Oct;30(10):2342-53

INSERM, U643, Nantes, France.

Besides their therapeutic benefit as cell source, neural stem/progenitor cells (NSPCs) exhibit immunosuppressive properties of great interest for modulating immune response in the central nervous system. To decipher the mechanisms of NSPC-mediated immunosuppression, activated T cells were exposed to NSPCs isolated from fetal rat brains. Analyses revealed that NSPCs inhibited T-cell proliferation and interferon-gamma production in a dose-dependent manner. A higher proportion of helper T cells (CD4+ T cells) was found in the presence of NSPCs, but analyses of FoxP3 population indicated that T-cell suppression was not secondary to an induction of suppressive regulatory T cells (FoxP3+ CD4+ CD25+). Conversely, induction of the high affinity interleukin-2 (IL-2) receptor (CD25) and the inability of IL-2 to rescue T-cell proliferation suggest that NSPCs display immunosuppressive activity without affecting T-cell activation. Cultures in Transwell chambers or addition of NSPC-conditioned medium to activated T cells indicated that part of the suppressive activity was not contact dependent. We therefore searched for soluble factors that mediate NSPC immunosuppression. We found that NSPCs express several immunosuppressive molecules, but the ability of these cells to inhibit T-cell proliferation was only counteracted by heme oxygenase (HO) inhibitors in association or not with nitric oxide synthase inhibitors. Taken together, our findings highlight a dynamic crosstalk between NSPCs and T lymphocytes and provide the first evidence of an implication of HO-1 in mediating the immunosuppressive effects of the NSPCs.
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http://dx.doi.org/10.1002/stem.1199DOI Listing
October 2012

Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications.

J Biol Chem 2012 Sep 10;287(40):33664-74. Epub 2012 Jul 10.

UMR INSERM 892-CNRS 6299, Centre de Recherche en Cancérologie Nantes-Angers, Nantes, France.

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.
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http://dx.doi.org/10.1074/jbc.M111.320028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460464PMC
September 2012

Pig neural cells derived from foetal mesencephalon as cell source for intracerebral xenotransplantation.

Methods Mol Biol 2012 ;885:233-43

INSERM, U643, Nantes, Cedex, France.

Intracerebral cell transplantation offers the possibility of replacing lost neurons in case of neurodegenerative disorders. To date, the best functional recovery for Parkinson's patients has been obtained using neuroblasts derived from human foetal mesencephalon, but the ethical and practical problems relative to the use of human foetal tissue lead to consideration of alternative sources of cells. In this regard, porcine neuroblasts appear as a valuable source as these cells are available in large quantity and programmed to extend long neurites as human neurons. However, the potential use of pig neural cells in the clinical setting depends on efficient and safe immunosuppression. So, most experimental work in this domain aims at developing immunosuppressive treatments specifically adapted to the central nervous system. In such perspective, transplantation of porcine mesencephalic neuroblasts into the striatum of the adult rat brain is of great interest. Indeed, rejection of intracerebral xenografts has been quite well described in rats, and graft survival can be easily monitored in a rat model of Parkinson's disease. In the present chapter, we describe the methods for isolating neuroblasts from foetal porcine mesencephalon as well as the technique of intracerebral transplantation in adult immunocompetent rats.
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http://dx.doi.org/10.1007/978-1-61779-845-0_14DOI Listing
September 2012

Neural stem/progenitor cells as a promising candidate for regenerative therapy of the central nervous system.

Front Cell Neurosci 2012 11;6:17. Epub 2012 Apr 11.

INSERM U643, Nantes, France.

Neural transplantation is a promising therapeutic strategy for neurodegenerative diseases and other disorders of the central nervous system (CNS) such as Parkinson and Huntington diseases, multiple sclerosis or stroke. Although cell replacement therapy already went through clinical trials for some of these diseases using fetal human neuroblasts, several significant limitations led to the search for alternative cell sources that would be more suitable for intracerebral transplantation.Taking into account logistical and ethical issues linked to the use of tissue derived from human fetuses, and the immunologically special status of the CNS allowing the occurrence of deleterious immune reactions, neural stem/progenitor cells (NSPCs) appear to be an interesting cell source candidate. In addition to their ability for replacing cell populations lost during the pathological events, NSPCs also display surprising therapeutic effects of neuroprotection and immunomodulation. A better knowledge of the mechanisms involved in these specific characteristics will hopefully lead in the future to a successful use of NSPCs in regenerative medicine for CNS disorders.
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http://dx.doi.org/10.3389/fncel.2012.00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323829PMC
October 2012

The immune molecule CD3zeta and its downstream effectors ZAP-70/Syk mediate ephrin signaling in neurons to regulate early neuritogenesis.

J Neurochem 2011 Nov 3;119(4):708-22. Epub 2011 Oct 3.

INSERM, UMR 643, Nantes, France.

Recent studies have highlighted the key role of the immune protein CD3ζ in the maturation of neuronal circuits in the CNS. Yet, the upstream signals that might recruit and activate CD3ζ in neurons are still unknown. In this study, we show that CD3ζ functions early in neuronal development and we identify ephrinA1-dependent EphA4 receptor activation as an upstream regulator of CD3ζ. When newly born neurons are still spherical, before neurite extension, we found a transient CD3ζ aggregation at the cell periphery matching the initiation site of the future neurite. This accumulation of CD3ζ correlated with a stimulatory effect on filopodia extension via a Rho-GEF Vav2 pathway and a repression of neurite outgrowth. Conversely, cultured neurons lacking CD3ζ isolated from CD3ζ(-/-) mice showed a decreased number of filopodia and an enhanced neurite number. Stimulation with ephrinA1 induces the translocation of both CD3ζ and its activated effector molecules, ZAP-70/Syk tyrosine kinases, to EphA4 receptor clusters. EphrinA1-induced growth cone collapse was abrogated in CD3ζ(-/-) neurons and was markedly reduced by ZAP-70/Syk inhibition. Moreover, ephrinA1-induced ZAP-70/Syk activation was inhibited in CD3ζ(-/-) neurons. Altogether, our data suggest that CD3ζ mediates the ZAP-70/Syk kinase activation triggered by ephrinA-activated pathway to regulate early neuronal morphogenesis.
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http://dx.doi.org/10.1111/j.1471-4159.2011.07469.xDOI Listing
November 2011

Ectopic expression of the immune adaptor protein CD3zeta in neural stem/progenitor cells disrupts cell-fate specification.

J Mol Neurosci 2012 Feb 2;46(2):431-41. Epub 2011 Aug 2.

UMR 643, INSERM, Nantes, 44000, France.

Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.
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http://dx.doi.org/10.1007/s12031-011-9607-2DOI Listing
February 2012

Effects of Human Alpha-Synuclein A53T-A30P Mutations on SVZ and Local Olfactory Bulb Cell Proliferation in a Transgenic Rat Model of Parkinson Disease.

Parkinsons Dis 2011 28;2011:987084. Epub 2011 Jun 28.

INSERM U 643, CHU Hôtel Dieu, 30 boulevard Jean Monnet, 44093 Nantes cedex 1, France.

A transgenic Sprague Dawley rat bearing the A30P and A53T α-synuclein (α-syn) human mutations under the control of the tyrosine hydroxylase promoter was generated in order to get a better understanding of the role of the human α-syn mutations on the neuropathological events involved in the progression of the Parkinson's disease (PD). This rat displayed olfactory deficits in the absence of motor impairments as observed in most early PD cases. In order to investigate the role of the mutated α-syn on cell proliferation, we focused on the subventricular zone (SVZ) and the olfactory bulbs (OB) as a change of the proliferation could affect OB function. The effect on OB dopaminergic innervation was investigated. The human α-syn co-localized in TH-positive OB neurons. No human α-syn was visualized in the SVZ. A significant increase in resident cell proliferation in the glomerular but not in the granular layers of the OB and in the SVZ was observed. TH innervation was significantly increased within the glomerular layer without an increase in the size of the glomeruli. Our rat could be a good model to investigate the role of human mutated α-syn on the development of olfactory deficits.
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http://dx.doi.org/10.4061/2011/987084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135113PMC
November 2011

Distinct roles of Bcl-2 and Bcl-Xl in the apoptosis of human bone marrow mesenchymal stem cells during differentiation.

PLoS One 2011 May 12;6(5):e19820. Epub 2011 May 12.

INSERM, UMR 892, Équipe Labellisée Ligue contre le Cancer, Nantes, France.

Background: Adult mesenchymal stem cells (MSCs) can be maintained over extended periods of time before activation and differentiation. Little is known about the programs that sustain the survival of these cells.

Principal Findings: Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in response to different cell death inducers. Conversely, the same inducers can readily induce apoptosis when hMSCs are engaged in the early stages of differentiation. The survival of undifferentiated cells is linked to the expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is expressed at similar levels in undifferentiated and differentiated hMSCs while Bcl-2 is expressed only in differentiated cells. In undifferentiated hMSCs, the down-regulation of Bcl-Xl is associated with an increased sensitivity to apoptosis while the ectopic expression of Bcl-2 induced apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in undifferentiated hMSCs.

Significance: In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an anti-apoptotic activity under all conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093403PMC
May 2011

Immunoregulatory properties of neural stem cells.

Immunotherapy 2011 Apr;3(4 Suppl):39-41

INSERM U643, CHU de Nantes, ITUN, Université de Nantes, Faculté de Médecine, Nantes, France.

Transplantation of neural cells provides an interesting form of therapy for certain CNS disorders. Although the brain has a special immune status, xenografts of fetal porcine neuroblasts are ultimately rejected after a lag of several weeks. Various strategies have been proposed to prevent this process. These include the design of transgenic pigs whose neurons have an increased immunosuppressive potential. An interesting alternative is provided by the use of neural stem/progenitor cells, which are multipotent cells found in the fetal or adult CNS. These cells are known to be poorly immunogenic. However, pig or rat neural stem/progenitor cells are highly immunosuppressive, as shown by their ability to block the proliferation of activated T lymphocytes. This effect is mediated by cell secreted factor(s), whose nature is discussed.
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http://dx.doi.org/10.2217/imt.11.49DOI Listing
April 2011

Intracerebral xenotransplantation: recent findings and perspectives for local immunosuppression.

Curr Opin Organ Transplant 2011 Apr;16(2):190-4

INSERM, UMR643, CHU de Nantes, Institut de Transplantation-Urologie-Néphrologie, ITUN, France.

Purpose Of Review: Cell therapy is a promising strategy for tissue repair in the central nervous system. In this perspective, several cell types are being considered, including allogenic neuroblasts, embryonic stem cells and induced pluripotent stem cells. The use of allogenic neuroblasts as cell source is limited by logistics and ethical problems whereas transplantation of the last two cell types is hampered by their propensity to generate tumour. In this context, transplantation of xenogeneic neural cells appears as an attractive approach for effective neuronal replacement in case of neurodegenerative disorders.

Recent Findings: With the emergence of embryonic and induced pluripotent stem cells as potential cell source in regenerative medicine, little attention has been paid to the possibility of transplanting xenogenic neural cells in the central nervous system. However, recent progress to circumvent the host immune response in the brain has raised encouraging perspectives for intracerebral xenotransplantation as restorative strategy.

Summary: To date, most of the immunosuppressive strategies designed for long-term survival of intracerebral neural transplants were based on systemic immunosuppression that has detrimental side-effects. The immunological status of the brain and the presence of the blood-brain barrier raise the possibility of local immunosuppression. This article provides an overview of the strategies recently developed to protect intracerebral neural transplants with special focus on local immunosuppression.
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http://dx.doi.org/10.1097/MOT.0b013e32834494b5DOI Listing
April 2011

In vitro analyses of the immunosuppressive properties of neural stem/progenitor cells using anti-CD3/CD28-activated T cells.

Methods Mol Biol 2011 ;677:233-43

INSERM UMR 643, Nantes, France.

Neural stem/progenitor cells (NSPCs) are multi-potent cells defined by their ability to self-renew and differentiate into cells of glial and neuronal lineage. Because of these properties, NSPCs have been proposed as therapeutic tools to replace lost neurons. Recent observations in animal models of immune-related diseases indicate that NSPCs display immunomodulatory properties that might be a great interest for cell therapy. In particular, transplantation of NSPCs might be very useful as local immunosuppressive agent to promote the long-term survival of neuronal xenotransplant in the brain. To study this possibility, we have analysed the impact of NSPCs on anti-CD3/CD28-activated T cells. In vitro analyses clearly show that porcine, rat, and mouse NSPCs inhibit the proliferation of activated T cells. This result raises new perspectives concerning the use of NSPCs in cell therapy.
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http://dx.doi.org/10.1007/978-1-60761-869-0_17DOI Listing
February 2011

Trophic and immunoregulatory properties of neural precursor cells: benefit for intracerebral transplantation.

Exp Neurol 2011 Jul 11;230(1):35-47. Epub 2010 May 11.

INSERM, UMR643, Nantes, France.

Intracerebral xenotransplantation of porcine fetal neuroblasts (pNB) is considered as an alternative to human neuroblasts for the treatment of neurodegenerative diseases. However, pNB are systematically rejected, even in an immunoprivileged site such as the brain. Within this context, neural stem/precursor cells (NSPC), which were suggested as exhibiting low immunogenicity, appeared as a useful source of xenogeneic cells. To determine the advantage of using porcine NSPC (pNSPC) in xenotransplantation, pNB and pNSPC were grafted into the striatum of rats without immunosuppression. At day 63, all the pNB were rejected while 40% of the rats transplanted with pNSPC exhibited large and healthy grafts with numerous pNF70-positive cells. The absence of inflammation at day 63 and the occasional presence of T cells in pNSPC grafts evoked a weak host immune response which might be partly due to the immunosuppressive properties of the transplanted cells. T cell proliferation assays confirmed such a hypothesis by revealing an inhibitory effect of pNSPC on T cells through a soluble factor. In addition to their immunosuppressive effect, in contrast to pNB, very few pNSPC differentiated into tyrosine hydroxylase-positive neurons but the cells triggered an intense innervation of the striatum by rat dopaminergic fibers coming from the substantia nigra. Further experiments will be required to optimize the use of pNSPC in regenerative medicine but here we show that their immunomodulatory and trophic activities might be of great interest for restorative strategies. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."
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http://dx.doi.org/10.1016/j.expneurol.2010.04.021DOI Listing
July 2011

Minocycline promotes long-term survival of neuronal transplant in the brain by inhibiting late microglial activation and T-cell recruitment.

Transplantation 2010 Apr;89(7):816-23

INSERM, UMR, Nantes, France.

Background: Cell therapy in the brain is limited by the requirement of high doses of immunosuppressors that have harmful side effects, and often, it cannot prevent the ultimate rejection of the transplanted cells. Alternative treatments that replace or enable a reduction in the doses of usual immunosuppressors have to be found. In this regard, minocycline shows potential as therapeutic agent. This drug crosses the blood-brain barrier, has good safety records, and exhibits strong antiinflammatory effects.

Methods: To study the impact of minocycline on the survival of intracerebral transplant, 400,000 porcine fetal neurons were transplanted into the striatum of rats treated daily with minocycline until sacrifice. Graft survival and immunologic reaction were evaluated by immunohistochemistry.

Results: In the control groups, all the grafts were rejected at day 63, whereas healthy grafts exhibiting tyrosine hydroxylase neurons were observed in 40% of the treated rats. The low immunoreactivity for ED1 and R73 in treated rats when compared with the control groups suggests that minocycline promotes long-term survival of neuronal xenograft by inhibiting microglial activation and T-cell recruitment.

Conclusions: Our present data provide the first evidence of an effect of minocycline on the host immune response after neuronal transplantation into the brain. This observation raises new perspectives concerning the use of minocycline and provides basis for the development of safe and efficient immunosuppressive protocols for intracerebral transplantation.
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http://dx.doi.org/10.1097/TP.0b013e3181cbe041DOI Listing
April 2010

Mesenchymal stem cells induce a weak immune response in the rat striatum after allo or xenotransplantation.

J Cell Mol Med 2009 Aug;13(8B):2547-58

INSERM UMR 643, Nantes cedex 1, France.

Mesenchymal stem cells (MSCs) have attracted attention for their potential use in regenerative medicine such as brain transplantation. As MSCs are considered to be hypoimmunogenic, transplanted MSCs should not trigger a strong host inflammatory response. To verify this hypothesis, we studied the brain immune response after transplantation of human or rat MSCs into the rat striatum and MSC fate at days 5, 14, 21 and 63 after transplantation. Flow cytometry analysis indicated that both MSCs express CD90 and human leucocyte antigen (MHC) class I, but no MHC class II molecules. They do not express CD45 or CD34 antigens. However, MSC phenotype varies with passage number. Human MSCs have mRNAs for interleukin (IL)-6, IL-8, IL-12, tumour necrosis factor (TNF)-alpha and TGF-beta(1), whereas rat MSCs express IL-6-, IL-10-, IL-12- and TGF-beta(1)-mRNAs. The quantification shows higher levels of mRNAs for the anti-inflammatory molecules IL-6 and TGF-beta(1) than for pro-inflammatory cytokines IL-8 and IL-12; ELISA analysis showed no IL-12 whereas TGF-beta(1) and IL-6 were detected. Transplant size did not significantly vary between 14 and 63 days after transplantation, indicating an absence of immune rejection of the grafts. Very few mast cells and moderate macrophage and microglial infiltrations, observed at day 5 remained stable until day 63 after transplantation in both rat and human MSC grafts. The observations of very few dendritic cells, T alphabeta-cells, and no T gammadelta-lymphocytes, all three being associated with Tp rejection in the brain, support the contention that MSCs are hypoimmunogenic. Our results suggest that MSCs are of great interest in regenerative medicine in a (xeno)transplantation setting.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00657.xDOI Listing
August 2009

AUF1 and Hu proteins in the developing rat brain: implication in the proliferation and differentiation of neural progenitors.

J Neurosci Res 2009 May;87(6):1296-309

INSERM U643, Nantes, France.

Posttranscriptional events such as RNA stabilization are important for cell differentiation, but little is known about the impact of AU-rich binding proteins (AUBPs) on the fate of neural cells. Expression of destabilizing AUBPs such as AUF1 and neuronal-specific stabilizing proteins such as HuB, HuC and HuD was therefore analyzed in the developing central nervous system. Real-time RT-PCR indicated a specific developmental pattern in the postnatal cerebellum, with a progressive down-regulation of AUF1 from P1, whereas HuB was strongly up-regulated at about P7. These changes were accompanied by a progressive increase in AUF1p45 and the disappearance of one HuB isoform from P15, suggesting particular roles for these AUBPs in the developing cerebellum. AUF1 was detected in the three main cerebellar layers, whereas Hu proteins were found only in postmitotic neurons. A role for Hu proteins in the early stages of neuronal differentiation is further supported by arrest of cell proliferation following induction of HuB or HuD expression in a neural stem cell line. The decrease in nestin expression suggest that HuD, but not HuB, favors the transition of neural progenitors into early neuroblasts, but other factors are most probably required for their full differentiation into neurons, insofar as GAP-43 was not detected in HuD-transfected cells. These data suggest critical roles for HuB at the very earliest stages of neuronal differentiation, such as cell cycle exit, and HuD might also be involved in the transition of neural progenitors into early neuroblasts. Taken together, the present results strengthen the importance of AUBPs in brain ontogenesis.
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http://dx.doi.org/10.1002/jnr.21957DOI Listing
May 2009

Cancer stem cells: beyond Koch's postulates.

Cancer Lett 2009 Jun 14;278(1):3-8. Epub 2008 Oct 14.

INSERM U646, F-49100, France.

Until the last century, infectious diseases were the leading cause of human mortality. Therefore, our current medical reasoning is profoundly influenced by views that originated from medical microbiology. The notion that cancer growth is sustained by a sub-population of particular cells, the cancer stem cells, is highly reminiscent of the germ theory of disease as exemplified by Koch's postulates in the XIXth century. However, accumulating data underscore the importance of cell-cell interactions and tumor environment. Hence it is essential to critically review the basic tenets of the cancer stem cell concept on the light of their relationships with Koch's postulates. Shifting the pathogenic element from a special cellular entity (cancer stem cell or microorganism) to a "pathogenic field" could be critical for curing both cancer and drug-resistant infectious diseases.
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http://dx.doi.org/10.1016/j.canlet.2008.09.006DOI Listing
June 2009

The signaling adaptor protein CD3zeta is a negative regulator of dendrite development in young neurons.

Mol Biol Cell 2008 Jun 26;19(6):2444-56. Epub 2008 Mar 26.

INSERM, U643, Nantes, F44000 France.

A novel idea is emergxsing that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3zeta, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3zeta is mainly expressed in neurons. Distribution of CD3zeta in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3zeta is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3zeta was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3zeta in cultured neurons or overexpression of a loss-of-function CD3zeta mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3zeta by a CD3zeta antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3zeta in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.
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http://dx.doi.org/10.1091/mbc.e07-09-0947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2397320PMC
June 2008

Cell surface antigens on rat neural progenitors and characterization of the CD3 (+)/CD3 (-) cell populations.

Differentiation 2006 Dec;74(9-10):530-41

INSERM, U643, Nantes F44000, France.

While the hematopoietic lineage has been extensively studied using cluster of differentiation (CD) antibodies, very few data are available on the extracellular epitopes expressed by rat neural progenitors (rNPC) and their derivatives. In the present study, we used flow cytometry to screen 47 cell surface antigens, initially known as immune markers. The quantitative analyses were performed on rat neurospheres and compared with primary cultures of astroglial cells or cerebellar neurons. Several antigens such as CD80 or CD86 were clearly undetectable while others, like CD26 or CD161, showed a weak expression. Interestingly, 10% and 15% of the cells were immunopositive for CD172a and CD200, two immunoglobulin superfamily members preferentially expressed by glial or neuronal cells, respectively. Over 40% of the cells were immunopositive for CD3, CD71, or MHCI. The biological significance of the latter markers in rNPC remains to be determined but analyses of the CD3(-)/CD3(+) populations isolated by magnetic cell separation revealed differences in their cell fate. Indeed, CD3(+) cells did not establish neurospheres and differentiated mostly into GFAP(+) cells while CD3(-) cells were able to generate neurospheres upon mitogen treatment and gave rise to GFAP(+), A2B5(+), Tuj-1(+), and RIP(+) cells under differentiating conditions. In contrast, CD71(-)/CD71(+) cells did not show any significant difference in their proliferating and differentiating potentials. Finally, it is worth noting that an subpopulation of cells in rat neurospheres exhibit an immunoreactivity against anti-CD25 (IL2 receptor) and anti-CD62L (L-selectin) antibodies. The results reveal particular surface antigen profiles, giving new perspectives on the properties of rat brain-derived cells.
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http://dx.doi.org/10.1111/j.1432-0436.2006.00098.xDOI Listing
December 2006