Il-10 overexpression does not synergize with the neuroprotective action of rgd-containing vectors after postnatal brain excitotoxicity, but modulates the main inflammatory cell responses



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IL-10 OVEREXPRESSION DOES NOT SYNERGIZE WITH THE NEUROPROTECTIVE ACTION OF RGD-CONTAINING VECTORS AFTER POSTNATAL BRAIN EXCITOTOXICITY, BUT MODULATES THE MAIN INFLAMMATORY CELL RESPONSES

Pau gonzalez, PhD (1, 5)*; Hugo Peluffo, PhD (2); Laia Acarin, PhD (1)*; Antonio Villaverde, PhD (3, 4); Berta Gonzalez, PhD (1) and Bernardo Castellano, PhD (1).



1Unit of Medical Histology, Department of Cell Biology, Physiology and Immunology and Neuroscience Institute, Autonomous University of Barcelona, Spain.

2Neurodegeneration Laboratory, Pasteur Institute of Montevideo and Department of Histology and Embryology, Faculty of Medicine, UDELAR, Uruguay.

3Institute of Biology and Biomedicine and Department of Genetics and Microbiology from the Autonomous University of Barcelona, Spain.

4Networking Biomedical Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain

5 Molecular Neurology Laboratory, National Paraplegic Hospital of Toledo, Spain

RUNNING TITLE: EFFECTS OF IL-10 OVEREXPRESSION IN IMMATURE BRAIN

*Corresponding authors.

Medical Histology, Torre M5, Department of Cell Biology, Physiology and Immunology, Autonomous University of Barcelona, 08193 Bellaterra (Barcelona), Spain. Tel: 34935811826, Fax: 34935812392, e-mail: laia.acarin@uab.cat, paug@sescam.jccm.es.


Supported by: BFU2005-02783 from the Ministry of Science and Innovation, Government of Spain and 061710 from the Marato TV3 Foundation to BG; BFU2008-04407/BFI from the Ministry of Science and Innovation, Government of Spain to BC and ACI2009-0910 from the Ministry of Science and Innovation, Government of Spain to AV.

ABSTRACT

Anti-inflammatory cytokines such as IL-10 have been used to modulate and terminate inflammation and provide neuroprotection. Recently, we reported that the modular recombinant transfection vector NLSCt is an efficient tool for transgene overexpression in vivo, which induces neuroprotection due to its RGD-mediated integrin interacting capacity. We here aimed to evaluate the putative synergic neuroprotective action exerted by IL-10 overexpression using NLSCt as transfection vector after an excitotoxic injury to the postnatal rat brain. For this purpose, lesion volume, neurodegeneration, astroglial and microglial responses, neutrophil infiltration and pro-inflammatory cytokine production were analyzed at several survival times after intracortical NMDA injection in postnatal day 9 rats followed by injection of NLSCt combined with the IL-10 gene, a control transgene or saline vehicle solution. Our results show no combined neuroprotective effect between RGD-interacting vectors and IL-10 gene therapy, instead IL-10 overexpression using NLSCt as transfection vector increased lesion volume and neuronal degeneration at 12 hours and 3 days post-lesion. In parallel, NLSCt/IL-10 treated animals displayed increased density of neutrophils and microglia/macrophages, and a reduced astroglial content of GFAP and vimentin. Moreover, NLSCt/IL-10 treated animals did not show any variation in interleukin-1β and tumour necrosis factor α expression but a slight increase in interleukin-6 content at 7 days pot-lesion. In conclusion, overexpression of IL-10 by using NLSCt transfection vector did not synergistically neuroprotect the excitotoxically damaged postnatal rat brain, but induced changes in the glial and inflammatory cell response.


Key words: IL-10, gene therapy, brain, postnatal, excitotoxicity
BACKGROUND

CNS inflammation is characterized by cellular responses including astroglial and microglial reactivity, leukocyte infiltration, and increased production of different inflammatory-related molecules such as pro-inflammatory cytokines, chemokines and free radical producing enzymes. Overall, these responses lead to an exacerbation in damage severity. To modulate and terminate the progression of inflammation, the expression of anti-inflammatory endogenous molecules is upregulated after CNS damage. Accordingly, interleukin-10 (IL-10), a cytokine with a strong anti-inflammatory nature (Moore et al. 2001; Strle et al. 2001), is upregulated in CNS pathologies such as multiple sclerosis (Hulshof et al. 2002) and in different experimental models of CNS injury including experimental autoimmune encephalomyelitis (EAE) (Jander et al. 1998; Ledeboer et al. 2003), middle cerebral artery occlusion (MCAO) (Zhai et al. 1997), traumatic brain injury (TBI) (Kamm et al. 2006) and a genetic model of Alzheimer disease (Apelt and Schliebs 2001). Noteworthy, as shown after MCAO, expression of pro-inflammatory molecules decreases when IL-10 is upregulated (Zhai et al. 1997). In agreement, the lack of IL-10 in knockout mice results in an elevated production of pro-inflammatory cytokines after endotoxic shock (Agnello et al. 2000), and several in vivo and in vitro experimental studies have reported the inhibitory role exerted by IL-10 administration on inflammatory processes such as astroglial and microglial reactivity (Balasingam et al. 1994; Balasingam and Yong 1996; Jackson et al. 2005; Mesples et al. 2003; Ooboshi et al. 2005; Pang et al. 2005), leukocyte infiltration (Knoblach and Faden 1998) and production of chemokines (Guo et al. 1998), pro-inflammatory cytokines (Benveniste et al. 1995; Knoblach and Faden 1998; Ledeboer et al. 2000; Molina-Holgado et al. 2001b; Ooboshi et al. 2005; Pousset et al. 2001; Sawada et al. 1999) and inflammatory-related enzymes (Molina-Holgado et al. 2002a; Molina-Holgado et al. 2002b; Molina-Holgado et al. 2001a). As a consequence, numerous experimental studies have focused on the neuroprotective potential of IL-10 administration during the course of different CNS pathologies achieving, in many cases, promising results (Brewer et al. 1999; Dietrich et al. 1999; Jackson et al. 2005; Knoblach and Faden 1998; Ooboshi et al. 2005; Spera et al. 1998).

However, only a few of the aforementioned studies have approached the actions exerted by IL-10 in the injured immature CNS. This lack of information becomes especially important when considering that the modulation of the inflammatory response associated to developmental brain damage constitutes a promising therapeutical approach (Acarin et al. 2001; Arvin et al. 2002; Cai et al. 2004; Chew et al. 2006; Cornette 2004; Dammann and Leviton 1997; Fan et al. 2005; Ferriero et al. 1996; Froen et al. 2002; Galasso et al. 2000; Gonzalez and Ferriero 2008; Hamada et al. 1994; Kadhim et al. 2001; Kremlev et al. 2007; Leviton and Dammann 2004; Lin et al. 2006; Mesples et al. 2003; Silverstein et al. 1997; Tahraoui et al. 2001; Tsuji et al. 2000), and that the immature brain displays distinct physiological and morphological features, as a consequence of its ongoing postnatal development, that determine its particular response to brain damage (Ferriero 2004; Vannucci and Hagberg 2004), including a higher susceptibility to inflammation-mediated damage and an exacerbation of the brain’s inflammatory response associated to injury (Dammann and Leviton 1997; Lawson and Perry 1995; Rezaie and Dean 2002). In this regard, we have recently shown that IL-10 and its receptor are upregulated in glial cells in the excitotoxically injured postnatal brain (Gonzalez et al. 2009), and that their increase temporally correlates with a decrease in the expression of pro-inflammatory molecules such as interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) (Acarin et al. 2000; Acarin et al. 2002), strongly supporting that IL-10 overexpression, alone or in combination with other therapies, may constitute an anti-inflammatory and putatively protective therapy for immature brain injury.

In order to modulate post-injury processes, we have demonstrated that the modular protein-based transfection vector NLSCt is an efficient tool for transgene overexpression after developmental excitotoxic damage (Peluffo et al. 2006; Peluffo et al. 2003). Each β-galactosidase subunit of the tetrameric NLSCt accommodates a polylysine tail with DNA condensing/attaching properties, a nuclear localization motif, and an integrin interacting domain obtained from the GH loop region of the foot-and-mouth disease virus, displaying a prototypical three-amino acid Arg-Gly-Asp (RGD) sequence, showing cell attachment and internalization properties (Aris and Villaverde 2003). As we previously shown, intracerebral injection of NLSCt combined with a transgene is able to induce the expression of the carried transgene after excitotoxic injury in neurons, microglia and astrocytes throughout the damaged tissue for at least 3 days post injection (Peluffo et al. 2003). Interestingly, NLSCt is not only effective as a transfection vector for in vivo gene delivery, but it also induces neuroprotection mediated by its RGD integrin interacting capacity (Peluffo et al. 2006; Peluffo et al. 2007). Taking advantage of this special feature of NLSCt, we have reported that the combined use of NLSCt together with the overexpression of transgenic Cu/Zn superoxide dismutase produced an additive response, increasing neuroprotection to excitotoxicity (Peluffo et al. 2005). As the neuroprotective effect of RGD-interaction was dependent on glial cells in vitro, and induced an early increase in microglial reactivity (Peluffo et al. 2006; Peluffo et al. 2007), we hypothesized that the introduction of a glial modulatory gene such as IL-10, would provide a more effective neuroprotective tool. Therefore, the aim of the present study was to evaluate the putative synergy between the NLSCt mediated neuroprotection and the potential anti-inflammatory and neuroprotective effects of IL-10 overexpression in the excitotoxically damaged postnatal rat brain.



MATERIALS AND METHODS

Animals and excitotoxic lesions.

All experimental animal work was conducted according to Spanish regulations following European Union directives. The ethical commission of the Autonomous University of Barcelona approved all experimental procedures. All efforts were made to minimize animal suffering in every step.

For this study, we used a total of 115 nine-day old (P9) Black Hooded Long Evans postnatal rats (15-20 gr) (Janvier, France) of both sexes (litter size was cut down to 10 on P0 and, in all cases, litters were composed by a similar number of males and females). Excitotoxic lesions were performed as previously described (Acarin et al. 1996; Acarin et al. 1997). Briefly, animals were placed in a stereotaxic frame adapted for newborns (Kopf) under isoflurane anesthesia (Veterinaria Esteve, Barcelona, Spain), the skull was opened using a drill and 0.15 μL of saline solution (0.9% NaCl, pH 7.4) containing 18.5 nmols of N-methyl-D-aspartate (NMDA) (Sigma, Steinheim, Germany) were injected into the right sensorimotor cortex at the level of the coronal suture, at 2 mm lateral to bregma and 0.5 mm of depth. After suture, pups were placed on a thermal pad and maintained at normothermia during 1 hour before being returned to the dam.

Treatment paradigm and experimental groups.

NLSCt was produced and purified from Escherichia coli as previously detailed (Aris and Villaverde 2003). Plasmid DNA containing the IL-10 gene (pORF5/mIL10) (Invitrogen, Paisley, UK) was purified from DH5α E. coli bacterial strain using “Wizard Plus Minipreps: DNA purification system” (Promega, Madison, WI) following supplier protocol. Complex formation between transfection vector and plasmidic DNA was induced by co-incubation of NLSCt (0.8 μg/μL) with the IL-10 plasmid (0.024 μg/μL) in saline solution at room temperature (RT) for one hour before injection.

Two hours after NMDA injection, pups were anaesthetized again and injected with 1 μL of NLSCt transfection vector (Peluffo et al. 2006) complexed to the plasmid containing the IL-10 gene, at the same stereotaxic coordinates used for NMDA administration. After suture, pups were placed again in a thermal pad and maintained at normothermia for 1 hour before being returned to the dam. Along this study, these animals will be referred as NMDA/NLSCt/IL-10. In addition, a set of NMDA lesioned animals were injected with either 1 μL of NLSCt complexed with a plasmid containing the GFP control gene (pEGFP-C1) (Clontech, Mountain View, CA) or 1 μL of vehicle saline solution. These animals were used as control groups and will be referred as NMDA/NLSCt/GFP and NMDA/SAL, respectively.

Experimental groups and sample processing.

According to the treatment received and the survival time, animals were distributed in different groups and subgroups as indicated in Table I. For histological and immunohistochemical analysis, rats were intracardially perfused with 4% paraformaldehyde (Sigma, Steinheim, Germany) in 0.1M phosphate buffer. Brains were removed, postfixed for 4 hours in the same fixative, cryoprotected in 30% sucrose, frozen with CO2 and stored at -80 ºC. Frozen brains were cut with the aid of a cryostat (Leica microsystems, Nussloch, Germany) and parallel 30 μm thick sections were obtained and stored either mounted on gelatin-coated slides or as free floating sections in Olmos antifreeze buffer at -20 ºC. For protein quantification by ELISA assays, pups were sacrificed by decapitation. Ipsilateral brain hemispheres were immediately removed, frozen with liquid nitrogen and stored at -80 ºC until homogenization in a potter homogenizer in ice-cold Tris-50mM/EDTA-1mM buffer containing a cocktail of protease (Roche, Manheim, Germany) and phosphatase inhibitors (Sigma, Steinheim, Germany). Finally, for IL-10 expression analysis by quantitative Real Time PCR, pups were killed by decapitation and ipsilateral brain hemispheres were immediately removed, frozen in liquid nitrogen and stored at -80 ºC until RNA isolation.



Histology and immunohistochemistry.

Nissl staining. For lesion volume quantification, a set of parallel sections mounted on gelatin-coated slides from each animal was stained following the Nissl method. Briefly, sections were incubated with 0.1% toluidine blue (Panreac, Barcelona, Spain) in Wallpole buffer at room temperature for 5 minutes and washed with distilled water. Sections were dehydrated, cleared in xylene (Panreac, Barcelona, Spain) and coverslipped with DPX (Panreac, Barcelona, Spain).

Fluoro-Jade B (FJB) staining. In order to visualize degenerating neurons, sections were processed for FJB labeling. Briefly, after hydration in decreasing graded ethanol, sections were rinsed in water and oxidized with 0.06% potassium permanganate (MnO4K) (Panreac, Barcelona, Spain) for 15 minutes. Then, sections were rinsed in distilled water and immersed for 20 minutes in 0.0004% FJB (Histo-Chem Inc, Jefferson, AK) plus 1% glacial acetic acid (Panreac, Barcelona, Spain) solution. Finally, sections were air dried, dehydrated and coverslipped with DPX (Panreac, Barcelona, Spain).

Tomato Lectin Histochemistry. To visualize microglia/macrophages, sections were processed for tomato lectin histochemistry (Acarin et al. 1994). Briefly, endogenous peroxidase was blocked by incubating slides for 10 minutes in 2% hydrogen peroxide (H2O2) (Sigma, Steinheim, Germany) diluted in 70% methanol (VWR Prolabo, Briare, France) solution. Sections were then rinsed and incubated for 2 h at 37 ºC in the biotinylated tomato lectin (TL) (Lycopersicon esculentum) (Sigma, Steinheim, Germany) in a 1:150 dilution. After washes, sections were incubated for 1 hour at RT with HRP-linked avidin (Dako, Glostrup, Denmark) diluted to 1:200. The peroxidase reaction product was visualized by incubating sections in 100 mL of Tris buffer containing 50 mg of 3-diaminobenzidine (DAB) (Sigma, Steinheim, Germany) and 33 μL H2O2 (Sigma, Steinheim, Germany). Finally, sections were dehydrated, cleared in xylene (Panreac, Barcelona, Spain) and coverslipped with DPX (Panreac, Barcelona, Spain).

Glial Fibrillary Acidic Protein (GFAP), vimentin and myeloperoxidase (MPO) immunohistochemistry. In order to visualize astrocytes, GFAP and vimentin immunohistochemistry were performed; whereas, MPO immunohistochemistry was carried out for neutrophil demonstration. Briefly, three sets of parallel sections mounted on gelatin-coated slides from each animal were processed for endogenous peroxidase blockade as detailed in the previous paragraph. Subsequently, sections were treated with buffer blocking (BB) containing 10% fetal calf serum (FCS) (Invitrogen, Paisley, UK) in Tris-buffered saline (pH 7.4) with 1% Triton X-100 (Sigma, Steinheim, Germany) for 1 h. Then, sections were incubated overnight at 4 ºC plus 1h at RT in one of the following primary antibodies: rabbit anti-GFAP (Dako, Glostrup, Denmark), mouse anti-vimentin (Dako, Glostrup, Denmark) or rabbit anti-MPO (Dako, Glostrup, Denmark) diluted to 1:500, 1:1000 and 1:400 in BB, respectively. After washes, sections were incubated for 1h in either HRP-linked anti-rabbit (GE Healthcare, Buckinghamshire, UK) or HRP-linked anti-mouse (GE Healthcare, Buckinghamshire, UK) secondary antibodies diluted to 1:200. The peroxidase reaction product was visualized using the DAB method as detailed before for TL histochemistry. Finally, sections were dehydrated, cleared in xylene (Panreac, Barcelona, Spain) and coverslipped with DPX (Panreac, Barcelona, Spain).

CD11b/phospho-histone H3 double immunohistochemistry. To evaluate microglia/macrophage proliferation, double immunofluorescence was carried out, in a set of free floating sections from a representative animal per experimental group and survival time, by combining CD11b (microglia/macrophage marker) and phospho-histone H3 (p-H3) (cell division marker). To accomplish that, sections were first incubated with mouse anti-CD11b primary antibody (Serotec, Oxford, UK) (1:1000) and subsequently with Cy2-conjugated anti-mouse secondary antibody (GE Healthcare, Buckinghamshire, UK) (1:1000). Samples were further processed by using rabbit anti-p-H3 primary antibody (Upstate, Temecula, CA) (1:3000) followed by incubation with Cy3-conjugated anti-rabbit (GE Healthcare, Buckinghamshire, UK) (1:1000). Finally, sections were dehydrated, cleared in xylene (Panreac, Barcelona, Spain) and coverslipped with DPX (Panreac, Barcelona, Spain).

Cytokine ELISA assays.

IL-1β, TNFα and IL-6 protein concentrations were evaluated in ipsilateral damaged hemispheres by using the following commercially available kits: Interleukin-1β [(r)IL-1β] rat ELISA system, Tumour Necrosis Factor Alpha [(r)TNFα] Rat Biotrak ELISA system and Interleukin-6 [(r)IL-6] rat ELISA system, according to the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). Total protein concentration of each sample was obtained by using the bicincholinic acid method (Smith et al. 1985).



RNA isolation, cDNA synthesis and Real Time PCR.

RNA was isolated from frozen ipsilateral damaged brain hemispheres by using the Total RNA isolation system Kit (Promega, Madison, WI). Concentration and purity of mRNA were determined by measuring absorbance at 260 and 280 nm and by electrophoresis. Moreover, in order to eliminate putative genomic DNA traces, further digestion with turbo-DNase (Applied Biosystems, Foster city, CA) was performed following the supplier protocol. DNA digestion was carried out at 37 ºC for 45 min followed by enzyme deactivation at 70 ºC for 15 min.

Two micrograms of mRNA were retrotranscribed, as described by the provider, by using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster city, CA) with random hexamers (Applied Biosystems, Foster city, CA) for transgenic IL-10 and with specific oligonucleotide primers for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (Table II). Retrotranscription was performed at 42 ºC for 45 min, followed by deactivation of the enzyme at 95 ºC for 5 min and 4 ºC for 5 min.

To perform GAPDH quantitative Real Time PCR, the SYBR Green PCR Core reagents kit was used (Applied Biosystems, Foster city, CA). Amplification of cDNA was carried out in MicroAmp Optical 384-wells Reaction Plates (Applied Biosystems, Foster city, CA) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster city, CA). The reaction mixture (10 μL) was composed of SYBR Green buffer, 3 mM of MgCl2 , 875 μM of dNTPs with dUTP, 0.3 U of AmpliTaq Gold, 0.12 U of Amperase UNG, 7.5 pmol of each primer (Table II), 12.5 ng of cDNA and nuclease-free H2O. The reaction conditions for cDNA amplification were 2 min at 50 ºC and 10 min at 95 ºC followed by 40 cycles of 15 seconds at 95 ºC and 1 min at 59 ºC. In order to carry out IL-10 Real Time PCR, we used the specific TaqMan Gene Expression Assay (Applied Biosystems, Foster city, CA) for murine IL-10, following supplier instructions. Amplification of cDNA was performed in MicroAmp Optical 96-wells Reaction Plates (Applied Biosystems, Foster city, CA) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster city, CA). In this case, cDNA was preamplified by initial 10 min at 95 ºC followed by 15 cycles of 15 seconds at 95 ºC and 1 min at 60 ºC. Finally, the amplification reaction was performed by initial 10 min at 95 ºC followed by 40 cycles of 15 seconds at 95 ºC and 1 min at 60 ºC. IL-10 mRNA expression was quantified with reference to the housekeeping gene GAPDH as: 2(Threshold cycle of target mRNA-Threshold cycle of GAPDH mRNA)



Lesion volume, densitometrical analysis and cell counting.

For lesion volume quantification, twenty two parallel Nissl stained sections (from bregma -1.8 mm to bregma +4.5 mm), separated by 300 μm and containing the whole lesion volume, were analyzed for each animal as previously described (Peluffo et al. 2006). In each section, lesioned area and total ipsilateral hemispheric area were measured by using analySIS 3.2 software (Soft Imaging System, Münster, Germany).

As NLSCt-induced transgene overexpression after postnatal excitotoxic injury is restricted to the lesioned nervous parenchyma (Peluffo et al. 2003), all densitometrical and cell count analysis were specifically performed in the lesioned areas. For densitometrical analysis, three sections from each animal corresponding to -0.3, 0 and +0.3 mm from bregma were selected and 20x images were taken at the center of the lesioned ipsilateral hemisphere as well as the corresponding contralateral non-lesioned area (Fig. 1). Images were analyzed by using analySIS 3.2 software (Soft Imaging System, Münster, Germany). The “immunoreactivity grade” was calculated as previously described (Acarin et al. 1997; Acarin et al. 1999b) for GFAP, vimentin and TL. In all cases, for each animal, values were obtained as the mean of three sections.

Quantification of microglia/macrophages and neutrophil density was performed in the same 20x micrographs (area = 0.18 mm2) used for densitometrical analysis (Fig. 1). To estimate the density of degenerating neurons, a set of two images/section (40x, area = 0.04 mm2) from the damaged tissue surrounding the central lesion core and from the same sections detailed above were used (Fig. 1). Images used for cell density evaluation were processed by using the tools for cell counting in AnalySIS 3.2 software (Soft Imaging System, Münster, Germany). Briefly, threshold was set qualitatively according to the immunohistochemical signal in contralateral control hemisphere, and it was maintained unchanged for all images. Subsequently, images were binarized and processed by the application of a particle separation filter. Finally, number of particles were measured in each image by using the analyze particles tool.



Statistical analysis.

In all cases, statistical analysis was performed using Statview 5.0.1 software and differences among groups were evaluated by means of one-way ANOVA followed by Fisher post-hoc test. All parameters were presented as mean values + SEM and p<0.05 was considered statistically significant.



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