A new role for interferon gamma in neural stem/precursor cell dysregulation
© Walter et al; licensee BioMed Central Ltd. 2011
Received: 6 October 2010
Accepted: 3 March 2011
Published: 3 March 2011
The identification of factors that compromise neurogenesis is aimed at improving stem cell-based approaches in the field of regenerative medicine. Interferon gamma (IFNγ) is a main pro-inflammatory cytokine and up-regulated during several neurological diseases. IFNγ is generally thought to beneficially enhance neurogenesis from fetal or adult neural stem/precursor cells (NSPCs).
We now provide direct evidence to the contrary that IFNγ induces a dysfunctional stage in a substantial portion of NSPC-derived progeny in vitro characterized by simultaneous expression of glial fibrillary acid protein (GFAP) and neuronal markers, an abnormal gene expression and a functional phenotype neither typical for neurons nor for mature astrocytes. Dysfunctional development of NSPCs under the influence of IFNγ was finally demonstrated by applying the microelectrode array technology. IFNγ exposure of NSPCs during an initial 7-day proliferation period prevented the subsequent adequate differentiation and formation of functional neuronal networks.
Our results show that immunocytochemical analyses of NSPC-derived progeny are not necessarily indicating the correct cellular phenotype specifically under inflammatory conditions and that simultaneous expression of neuronal and glial markers rather point to cellular dysregulation. We hypothesize that inhibiting the impact of IFNγ on NSPCs during neurological diseases might contribute to effective neurogenesis and regeneration.
Neural stem/precursor cells (NSPCs) may be useful as an endogenous or transplantable source of newly generated neural cells, which can replace lost or diseased neurons within the central nervous system (CNS) . A prerequisite for this is an appropriate functional differentiation of immature neural cells into electrophysiologically active neurons. As nearly all CNS diseases involve acute and chronic inflammatory processes , it is crucial to understand NSPC development under inflammatory conditions to better realize their full potential. IFNγ is a key inflammatory cytokine, mainly produced by cytotoxic CD8+ T-cells and natural killer cells in the course of neurological diseases like cerebral traumata , stroke  or multiple sclerosis . Beside the observation that IFNγ-activated microglial cells induce neurogenesis , IFNγ has also been reported to exert beneficial, pro-neurogenic effects on NSPC development in vitro and in vivo in a number of recent publications independently of microglial cells [7–9]. However, a hint that IFNγ might be involved in potentially harmful developmental dysregulation was detected in a number of reports [10–12] and from its tumor-initiating role, since embryonic mice over-expressing IFNγ develop medulloblastomas , indicating that IFNγ may also be involved in malignant transformation of neural precursor cells.
In the present study, we demonstrated that IFNγ induces an abnormal immunocytochemical phenotype in NSPCs with simultaneous expression of neuronal and glial markers. Furthermore, IFNγ led to a dysregulated gene expression as well as dysfunctional electrophysiological properties. Additionally, we finally present evidence that IFNγ exposure to NSPCs during an initial 7-day proliferation period dramatically impairs the subsequent development of functional neuronal networks as recorded by the microelectrode array technology. Our data clearly indicate that IFNγ compromises neurogenesis. Thus, its role during inflammatory processes should be reassessed and IFNγ suppression during brain pathology possibly supports functional neurogenesis.
IFNγ receptors 1 and 2 are expressed in NSPCs and their differentiated progeny
IFNγ reduces the population extent of NSPCs
IFNγ induces an abnormal phenotype in NSPCs
IFNγ induces an abnormal down-stream signaling in NSPCs
GFAP+/βIII-tubulin+ cells exhibit non-neuronal and non-astrocytic functional properties
IFNγ treatment impaired the formation of in vitro-functional neural networks
Our results shed new light on the effects of IFNγ on NSPCs. Until now, IFNγ-related up-regulation of βIII-tubulin was interpreted as a beneficial enhancement of neurogenesis [7–9]. The present study disproves this view and shows that IFNγ instead promotes an abnormal NSPC-derived cellular phenotype that does not relate to classical neurons or astrocytes and that appears to be dysregulated in terms of functional and molecular properties. IFNγ treatment leads to the expression of both, class III βtubulin and GFAP in ~40% of NSPC which is abnormal and, even after differentiation, not linked to mature neuronal or astrocytic electrophysiological function. Class III βtubulin isotype is usually considered specific for post-mitotic neurons, and such aberrant expression has so far only been noted in gliomas [15, 16] or dysregulated tumorigenic neural stem cells [17, 18]. Walton and colleagues even report some unusual co-expression of βIII-tubulin and GFAP in tumorigenic neural stem cells, a phenomenon similar to that detected here after IFNγ treatment of regular NSPCs. The aspect of IFNγ-mediated NSPC dysregulation is further substantiated in the present report by an up-regulation of SHH which is paralleled by down-regulation of Gli1 which has been reported to be consistently up-regulated in the course of SHH signaling . As expression patterns of neurogenic niche morphogenes like SHH or Gli1 are generally tightly regulated during CNS development, its disturbance points to misguided development or again tumorigenesis [13, 19]. Thus, for the first time, we directly illustrated a possible link between IFNγ, NSPCs and cellular abnormalities similar to that observed in tumor cells strongly supporting the view that inflammation might be involved in tumor generation via neural stem cells. Additionally, the IFNγ-related down-regulation of iNOS in NSPC cultures is untypical as it is known that IFNγ normally induces iNOS . Our electrophysiological findings illustrate the importance of an additional functional control of morphological/immunocytochemical observations as the up-regulation of βIII-tubulin in differentiated NSPC-derived cells, which was interpreted as enhanced neurogenesis in different studies [7, 9], was not paralleled by neuronal electrophysiological behavior. Further, the increase in GFAP-/βIII-tubulin+ neurons after IFNγ treatment of proliferating cultures was not significant in the present study and after differentiation under the impact of IFNγ we even found significantly less GFAP-/βIII-tubulin+ neurons. Interestingly, a similar observation was described previously . We found that those βIII-tubulin expressing cells that significantly increased in numbers after IFNγ treatment of proliferating or differentiating cultures were also GFAP positive and exhibited electrophysiological properties that were neither typical for mature astrocytes nor for neurons. We demonstrated this by careful correlating electrophysiological data of patched cells with their immunocytochemical phenotype. These molecular and functional IFNγ effects on NSPCs indicate a profoundly compromised cell function or, alternatively, a new IFNγ-induced NSPC-derived neural cell of unknown function. Interestingly, ectopic expression of IFNγ during early stages of CNS development induces medulloblastomas via SHH overexpression  pointing towards a general dysregulating effect of IFNγ on NSPCs during development or disease.
To investigate functional neural development under controlled conditions, with and without IFNγ-treatment, we electrophysiologically measured the development of functional neuronal networks starting from ES cell-derived immature neural precursor cell cultures. Usually, network activity progressively develops over time as a result of a complex interaction of a multitude of factors that converge to an integrated functional entity . It depends on efficient synapse formation and function of an entire neuronal population. If using immature neural precursor populations as developmental starting point, basic aspects of functional neural development can be measured. In contrast, mature ES cell-derived functional neuronal networks can be used to detect acute functional consequences due to changes in extracellular composition. These investigations then affect already active neuronal networks. For instance, they showed to reversibly alter their network function under the influence of different cerebrospinal fluid specimens . We chose a paradigm in which the influence of IFNγ selectively affected the initial proliferation period of cultures that were subsequently held under normal differentiating conditions. IFNγ-treated cultures showed a significantly impaired development of neuronal network function, impressively pointing to an IFNγ-related, profoundly altered functional development of neural precursor populations.
Thus, we speculate that abnormally high IFNγ production during development and CNS diseases might impair functional neuronal development in fetal neurogenesis or adult regeneration and propose to inhibit IFNγ effects on NSPCs as a means to effectively support their developmental and regenerative potential.
Materials and methods
Neurospheres were generated from fourteen-day-old wild-type C57BL/6J mouse embryos. Ganglionic eminences were removed, mechanically dissociated and seeded in DMEM/F12 culture medium (1:1; Invitrogen, Karlsruhe, Germany) containing 0.6% Glucose (Sigma-Aldrich, Hamburg, Germany), glutamine (2 mM; Invitrogen), sodium bicarbonate (3 mM; Invitrogen), Hepes buffer (5 mM; Invitrogen) and B27 (20 μl per ml; Invitrogen). For generation and expansion of neurosphere cells, epidermal growth factor (EGF) (Tebu-bio, Le Perray en Yvelines Cedex, France) and basic fibroblast growth factor-2 (FGF-2) (Tebu-bio) were added to a final concentration of 20 ng per ml each.
Generation of embryonic stem cell-derived neural stem cells
Undifferentiated ES cells (SV-129, ATCC, Millipore, Billerica, USA) were grown under feeder-deprived conditions in the presence of 1000 U/ml leukemia inhibitory factor (LIF, Millipore) and 20% fetal bovine serum (FBS, HyClone, Thermo Fisher Scientific, Schwerte, Germany) in ES cell medium described elsewhere . Neural differentiation of immature ES cells into neural stem cell (NS cells) was performed according to modified protocols [22, 25].
IFNγ treatment and immunocytochemistry
For immunocytochemistry, neurosphere cells or ES cell-derived NS cells were dissociated to a single cell suspension and plated on poly-L-ornithine (PLO; 0.001%; Sigma-Aldrich) and fibronectin (5 μg/ml; Tebu-bio) coated cover slips (VWR International, Darmstadt, Germany) at a density of 50 × 103 cells per ml. After 3 days under the influence of EGF and FGF-2 (20 ng/ml both Tebu-bio), cells were assigned to the different experimental groups. To verify the marker expression of undifferentiated (proliferating) neural populations under control or IFNγ treated conditions, cultures were kept under the influence of EGF/FGF-2 without or with IFNγ (1000 U/ml; Millipore) until fixation after further 3 days (NSPC-p -IFNγ/+IFNγ). To verify cell-type specific marker expression in differentiated cultures, growth factors were withdrawn and then, cells were treated for 7 days without or with IFNγ until fixation (NSPC-d -IFNγ/+IFNγ). For control experiments, only phosphate-buffered saline solution (PBS; 1X; Invitrogen) was added to the medium. Primary antibodies used at 4°C overnight were monoclonal mouse antibodies to 5-bromo-2-deoxyuridine (BrdU; 1:1000, Sigma-Aldrich), βIII-tubulin (Tuj1; 1:500; R&D Systems, Minneapolis, USA or 1:800, Abcam, Cambridge, UK), Map2a-c (1:2000; Sigma-Aldrich), Sox2 (1:50; R&D Systems), IFNγ-R1 (1:500; Santa Cruz Biotechnology) and beta Actin (1:100; Millipore) and polyclonal rabbit antibodies to glial fibrillaric acid protein (GFAP) (1:500; Dako, Hamburg, Germany or 1:1000; Abcam), caspase (1:100; Cell Signaling), IFNγ-R2 (1:500; Santa Cruz Biotechnology) and nestin (1:200; Covance). BrdU labeling is described elsewhere (Wellen et al., 2009). For detection of primary antibodies, fluoresceine-isothiocyanate (FITC; 1:500; Millipore) and indocarbocyanine (Cy3; 1:800; or Cy5; 1:200; Millipore) coupled secondary antibodies were used. For negative controls, primary antibodies were omitted in each experiment. To measure the total population of cells, Dapi positive cell nuclei were counted. On every cover slip, at least 100 cells were counted.
To analyze the population extent of NSPCs, the optical density , indicative of conversion of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) into formazan crystals which takes place in live cells only, was determined after IFNγ treatment at decreasing concentrations as indicated. An OD value of 0.5 represents approximately 50,000, and an OD value of 1.0 represents approximately 100,000 live NSPCs. The population extent was measured after 48 hours of IFNγ treatment as indicated.
For detection of caspase 3/7 activity after IFNγ treatment, we used the Caspase-Glo 3/7 assay (Promega, Madison, USA). Proliferating cultures were treated with decreasing concentrations of IFNγ as indicated. Adding the assay components to cultivated cells leads to cell lysis and release of caspase 3/7. Caspase 3/7 is capable of cleaving a tetrapeptide sequence substrate; this is dismantled by luciferase which is a component of the assay. The resulting light emission is then a measure of caspase activity.
Quantitative real-time PCR
Somatic whole-cell patch-clamp recordings were carried out using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) coupled to a personal computer via a digidata 1322A interface (Molecular Devices). Data were acquired at 10 kHz using PClamp 8.2 software (Molecular Devices). Patch pipettes were pulled from borosilicate glass (Hilgenberg, Waldkappel, Germany) and had a resistance of 3-6 MΩ when filled with intracellular solution containing (in mM): 120 K-MeSO3, 32 KCl, 10 HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), 4 NaCl, 4 Mg-ATP and 0.4 Na-GTP, 1 Alexa Fluor 350 (Molecular Probes/Invitrogen), pH 7.30 (calculated liquid junction potential: 12.5 mV). Cells were held at membrane potentials of -70 mV. To separate passive conductances from voltage-gated currents, online leak subtraction (P/4) was performed. Extracellular solution during patch-clamp experiments contained in mM: 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose, bubbled with 95% O2 and 5% CO2 to result in a pH of 7.4. Patch-clamp data were processed and analyzed using IGOR Pro-Software (WaveMetrics, Inc., Lake Oswego, OR). After the recordings, patch-pipettes were carefully withdrawn and coverslips were transferred into 4% paraformaldehyde for 20 minutes at room temperature. Thereafter coverslips were kept in PBS (Invitrogen) at 4 °C until they were processed for GFAP and βtubulin immunocytochemistry. By means of fluorescence at 350 nm electrophysiologically recorded cells were identified and assigned either to GFAP+/βIII-tubulin+ or GFAP+/βIII-tubulin- cells.
Microelectrode array recordings
For microelectrode array (MEA) recordings, 5 to 10 neural precursor cell-enriched, serum-free, floating embryoid body-like aggregates (nSFEBs)  were seeded on poly-D-lysine (PDL, 15 μg/ml, Sigma-Aldrich, Germany) and laminin (15 μg/ml, Sigma-Aldrich, Germany) coated MEAs with a square grid of 60 planar Ti/TiN electrodes (30-μm diameter, 200-μm spacing) and an input impedance of <50 kÙ according to the specifications of the manufacturer (Multi Channel Systems, Reutlingen, Germany). Signals from all 60 electrodes were simultaneously sampled at 25 kHz, visualized and stored using the standard software MC Rack provided by Multi Channel Systems. Spike and burst detection was performed off-line by custom-built software (Result, Düsseldorf, Germany). nSFEBs were kept for 7 days after plating under the influence of FGF-2 (20 ng/ml, PeproTech) only (IFN-γ - group) or together with IFN-γ (1000 U/ml; IFN-γ + group). After 7 days, FGF-2 and IFN-γ were removed from the medium to induce terminal differentiation. For long-term culture, ES cell-derived neuronal networks were kept in DMEM/F12 (Gibco) supplemented with N2, B27 and Glutamax (all Invitrogen). MEA recordings were performed at the indicated time points.
Experiments were repeated with independent cultures at least three times in triplicate each. The resulting data sets were statistically analyzed und illustrated using the GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA, USA, 2003) software. For approval of statistical significance between groups a two-tailed t-test was performed. P values < 0.05 were considered to indicate significant differences. For comparison of functional neuronal network development, slopes of linear regressions were calculated with GraphPad Prism 4 and p- and F-values were given.
We are grateful to Sabine Hamm and Christine Holland for excellent technical assistance, animal care and laboratory support.
- Bjorklund A, Lindvall O: Cell replacement therapies for central nervous system disorders. Nat Neurosci. 2000, 3: 537-544. 10.1038/75705.PubMedView ArticleGoogle Scholar
- Lucas SM, Rothwell NJ, Gibson RM: The role of inflammation in CNS injury and disease. Br J Pharmacol. 2006, 147 (Suppl 1): S232-240.PubMedPubMed CentralGoogle Scholar
- Schmitz T, Chew LJ: Cytokines and myelination in the central nervous system. ScientificWorldJournal. 2008, 8: 1119-1147. 10.1100/tsw.2008.140.PubMedPubMed CentralView ArticleGoogle Scholar
- Liesz A, Suri-Payer E, Veltkamp C, Doerr H, Sommer C, Rivest S, Giese T, Veltkamp R: Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med. 2009, 15: 192-199. 10.1038/nm.1927.PubMedView ArticleGoogle Scholar
- Lees JR, Golumbek PT, Sim J, Dorsey D, Russell JH: Regional CNS responses to IFN-gamma determine lesion localization patterns during EAE pathogenesis. J Exp Med. 2008, 205: 2633-2642. 10.1084/jem.20080155.PubMedPubMed CentralView ArticleGoogle Scholar
- Butovsky O, Ziv Y, Schwartz A, Landa G, Talpalar AE, Pluchino S, Martino G, Schwartz M: Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells. Mol Cell Neurosci. 2006, 31: 149-160. 10.1016/j.mcn.2005.10.006.PubMedView ArticleGoogle Scholar
- Whitney NP, Eidem TM, Peng H, Huang Y, Zheng JC: Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009, 108: 1343-1359. 10.1111/j.1471-4159.2009.05886.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Baron R, Nemirovsky A, Harpaz I, Cohen H, Owens T, Monsonego A: IFN-gamma enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease. Faseb J. 2008, 22: 2843-2852. 10.1096/fj.08-105866.PubMedView ArticleGoogle Scholar
- Johansson S, Price J, Modo M: Effect of inflammatory cytokines on major histocompatibility complex expression and differentiation of human neural stem/progenitor cells. Stem Cells. 2008, 26: 2444-2454. 10.1634/stemcells.2008-0116.PubMedView ArticleGoogle Scholar
- Wang J, Lin W, Popko B, Campbell IL: Inducible production of interferon-gamma in the developing brain causes cerebellar dysplasia with activation of the Sonic hedgehog pathway. Mol Cell Neurosci. 2004, 27: 489-496. 10.1016/j.mcn.2004.08.004.PubMedView ArticleGoogle Scholar
- LaFerla FM, Sugarman MC, Lane TE, Leissring MA: Regional hypomyelination and dysplasia in transgenic mice with astrocyte-directed expression of interferon-gamma. J Mol Neurosci. 2000, 15: 45-59. 10.1385/JMN:15:1:45.PubMedView ArticleGoogle Scholar
- Kim IJ, Beck HN, Lein PJ, Higgins D: Interferon gamma induces retrograde dendritic retraction and inhibits synapse formation. J Neurosci. 2002, 22: 4530-4539.PubMedGoogle Scholar
- Lin W, Kemper A, McCarthy KD, Pytel P, Wang JP, Campbell IL, Utset MF, Popko B: Interferon-gamma induced medulloblastoma in the developing cerebellum. J Neurosci. 2004, 24: 10074-10083. 10.1523/JNEUROSCI.2604-04.2004.PubMedView ArticleGoogle Scholar
- Zhou M, Schools GP, Kimelberg HK: Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive. J Neurophysiol. 2006, 95: 134-143. 10.1152/jn.00570.2005.PubMedView ArticleGoogle Scholar
- Ignatova TN, Kukekov VG, Laywell ED, Suslov ON, Vrionis FD, Steindler DA: Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia. 2002, 39: 193-206. 10.1002/glia.10094.PubMedView ArticleGoogle Scholar
- Katsetos CD, Del Valle L, Geddes JF, Assimakopoulou M, Legido A, Boyd JC, Balin B, Parikh NA, Maraziotis T, de Chadarevian JP, et al: Aberrant localization of the neuronal class III beta-tubulin in astrocytomas. Arch Pathol Lab Med. 2001, 125: 613-624.PubMedGoogle Scholar
- Walton NM, Snyder GE, Park D, Kobeissy F, Scheffler B, Steindler DA: Gliotypic neural stem cells transiently adopt tumorigenic properties during normal differentiation. Stem Cells. 2009, 27: 280-289. 10.1634/stemcells.2008-0842.PubMedPubMed CentralView ArticleGoogle Scholar
- Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB: Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003, 63: 5821-5828.PubMedGoogle Scholar
- Stecca B, Ruiz i Altaba A: Brain as a paradigm of organ growth: Hedgehog-Gli signaling in neural stem cells and brain tumors. J Neurobiol. 2005, 64: 476-490. 10.1002/neu.20160.PubMedView ArticleGoogle Scholar
- Bach EA, Aguet M, Schreiber RD: The IFN gamma receptor: a paradigm for cytokine receptor signaling. Annu Rev Immunol. 1997, 15: 563-591. 10.1146/annurev.immunol.15.1.563.PubMedView ArticleGoogle Scholar
- Wang Y, Imitola J, Rasmussen S, O'Connor KC, Khoury SJ: Paradoxical dysregulation of the neural stem cell pathway sonic hedgehog-Gli1 in autoimmune encephalomyelitis and multiple sclerosis. Ann Neurol. 2008, 64: 417-427. 10.1002/ana.21457.PubMedPubMed CentralView ArticleGoogle Scholar
- Illes S, Theiss S, Hartung HP, Siebler M, Dihne M: Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations. BMC Neurosci. 2009, 10: 93-10.1186/1471-2202-10-93.PubMedPubMed CentralView ArticleGoogle Scholar
- Otto F, Illes S, Opatz J, Laryea M, Theiss S, Hartung HP, Schnitzler A, Siebler M, Dihne M: Cerebrospinal fluid of brain trauma patients inhibits in vitro neuronal network function via NMDA receptors. Ann Neurol. 2009, 66: 546-555. 10.1002/ana.21808.PubMedView ArticleGoogle Scholar
- Okabe S, Forsberg-Nilsson K, Spiro AC, Segal M, McKay RD: Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech Dev. 1996, 59: 89-102. 10.1016/0925-4773(96)00572-2.PubMedView ArticleGoogle Scholar
- Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A: Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol. 2005, 3: e283-10.1371/journal.pbio.0030283.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.