Brain, Behavior, and Immunity 37 (2014) 248–259

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Effector molecules released by Th1 but not Th17 cells drive an M1 response in microglia Chittappen K. Prajeeth a, Kirsten Löhr b, Stefan Floess b, Julian Zimmermann e, Reiner Ulrich c, Viktoria Gudi a, Andreas Beineke c, Wolfgang Baumgärtner c,d, Marcus Müller e, Jochen Huehn b,1, Martin Stangel a,d,⇑,1 a

Clinical Neuroimmunology and Neurochemistry, Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany d Center of Systems Neuroscience, Hannover, Germany e Department of Neurology, Universitätsklinikum Bonn, Bonn, Germany b c

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Article history: Received 3 November 2013 Received in revised form 23 December 2013 Accepted 2 January 2014 Available online 9 January 2014 Keywords: Microglia Th1 Th17

a b s t r a c t Microglia act as sensors of inflammation in the central nervous system (CNS) and respond to many stimuli. Other key players in neuroinflammatory diseases are CD4+ T helper cell (Th) subsets that characteristically secrete IFN-c (Th1) or IL-17 (Th17). However, the potential of a distinct cytokine milieu generated by these effector T cell subsets to modulate microglial phenotype and function is poorly understood. We therefore investigated the ability of factors secreted by Th1 and Th17 cells to induce microglial activation. In vitro experiments wherein microglia were cultured in the presence of supernatants derived from polarized Th1 or Th17 cultures, revealed that Th1-associated factors could directly activate and trigger a proinflammatory M1-type gene expression profile in microglia that was cell–cell contact independent, whereas Th17 cells or its associated factors did not have any direct influence on microglia. To assess the effects of the key Th17 effector cytokine IL-17A in vivo we used transgenic mice in which IL-17A is specifically expressed in astrocytes. Flow cytometric and histological analysis revealed only subtle changes in the phenotype of microglia suggesting only minimal effects of constitutively produced IL17A on microglia in vivo. Neither IL-23 signaling nor addition of GM-CSF, a recently described effector molecule of Th17 cells, changed the incapacity of Th17 cells to activate microglia. These findings demonstrate a potent effect of Th1 cells on microglia, however, the mechanism of how Th17 cells achieve their effect in CNS inflammation remains unclear. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Neuroinflammation is a consequence of a crosstalk between resident immune cells of the CNS and cells infiltrating from the periphery (Ransohoff and Brown, 2012; Ransohoff and Engelhardt, 2012). Microglia are crucial in maintaining the integrity of the CNS, as they constantly sample the microenvironment and act as sensors of pathological conditions. The activation state of microglia strikes a balance between tissue damage and repair. In steady state, the resting microglia express low levels of major histocompatibility complex (MHC) class II molecules and co-stimulatory molecules on their surface and are mostly involved in maintenance ⇑ Corresponding author at: Clinical Neuroimmunology and Neurochemistry, Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: +49 511 532 6676; fax: +49 511 532 3115. E-mail address: [email protected] (M. Stangel). 1 Shared senior authors. 0889-1591/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2014.01.001

of homeostasis in CNS (Aguzzi et al., 2013; Goldmann and Prinz, 2013; Tambuyzer et al., 2009). However, an inflammatory insult leads to activation of microglia and triggers a proinflammatory cascade that can cause tissue damage. Reports suggest that microglial activation is a key event in most of the neuroinflammatory and neurodegenerative disorders (Bhasin et al., 2007; Heppner et al., 2005). It is well understood that IFN-c-producing Th1 cells and IL-17producing Th17 cells play a crucial role in the pathology of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) and similar neurodegenerative disorders (Goverman, 2009; Pierson et al., 2012). The individual contribution of Th1 and Th17 cells in CNS pathologies is highly debatable but a consensus arises from the fact that both subsets can mediate EAE although with varying degree of severity when adoptively transferred into mice (Domingues et al., 2010; Gocke et al., 2007; Lee et al., 2012; Yang et al., 2009). Characteristically, these subsets have distinct cytokine secretory profiles as their differentiation is controlled by mutually

C.K. Prajeeth et al. / Brain, Behavior, and Immunity 37 (2014) 248–259

exclusive signaling molecules and transcription factors (Sallusto et al., 2012; Stockinger and Veldhoen, 2007; Wilson et al., 2009). Further evidence suggests that both Th1 and Th17 cells can interact with microglia and activate them (McQuillan et al., 2010; Murphy et al., 2010). However, the effectiveness of the effector molecules secreted by Th1 and Th17 cells in activating and inducing a robust inflammatory response in microglia is only poorly understood. Several methods have been published describing the polarizing conditions used to generate Th1 and Th17 cells from naïve CD4+ T cells (Carlson et al., 2009; Nurieva et al., 2009). In this study, we employed the supernatants generated from in vitro differentiated highly pure Th1 and Th17 cells and compared their effects on microglia harvested from mixed glial cultures. Our findings reveal that Th1-, but not Th17-derived supernatants were effective in microglial activation as they significantly upregulated the expression of co-stimulatory molecules on microglia and bestow them with a pro-inflammatory phenotype. Interestingly, key Th-17 effector molecules, IL-17A and GM-CSF, had no direct influence on microglia in vitro. Even constitutive long-term expression of IL-17A in the CNS of transgenic mice led to only subtle changes in microglia in vivo. 2. Materials and methods 2.1. Mice C57BL/6 male mice were obtained from Charles River (Sulzfeld, Germany) or from Janvier and housed under specific-pathogenfree conditions in the central animal facility of Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany, Hannover Medical School (MHH), Germany and at Institute of pathology, University of Veterinary Medicine (TiHo), Hannover, Germany. Generation of the GF/IL17 transgenic mice where IL17 is specifically expressed under GFAP promoter has been previously described (Zimmermann et al., 2013). For generation of GF/IL17 mice, Il17a cDNA fragment was cloned into a GFAP expression vector containing a human growth hormone polyadenylation signal sequence downstream of the insert. The resulting fusion gene construct was microinjected into the germline of (C57Bl/6  C3H/HeN) F1 mice. Genotyping of the animals was accomplished by PCR analysis of genomic tail DNA using primers targeted at the human growth hormone sequence and the Il17a sequence included in the transgene construct. Hemizygote transgenic founder mice were backcrossed to the C57BL/6 background for at least 8 generations before experiments were performed. Transgene negative mice served as wild-type littermate controls. GF/IL17 transgenic mice were maintained under specific-pathogen-free conditions in the closed breeding colony of the University Hospital of Muenster, Germany. All research and animal care procedures were approved by the Review Board of the care for Animals Subjects of the district government (Lower Saxony, Germany) and performed according to international guidelines on the use of laboratory animals (Nicklas et al., 2002). 2.2. Antibodies and reagents Antibodies specific for mouse, anti-CD3 PerCp Cy5.5 (clone: 145-2C11), anti-CD4 FITC (clone: RM 4.5), anti-CD25 APC (clone: PC61), and anti-CD40 PE and APC (clone: 1C10) were purchased from eBioscience, Frankfurt, Germany. Antibodies to anti-IFN-c PE (clone: XMG1.2), anti-IL-17A APC (clone: TC11-18H10.1), antiCD11b PE and PerCp (clone: M1/70), anti-I-A/I-E FITC (clone: M5/ 114.15.2) and anti-CD86 FITC (clone: GL-1) were purchased from Biolegend, San Diego, CA. Rat anti-mouse IgG2a j PE, and FITC

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(clone: eBR2A) and IgG2b j FITC (clone: eB149/10H5) were purchased from eBioscience, Frankfurt, Germany. Unconjugated antiCD3 (clone: 145-2C11), anti-CD28 (clone: 37.51), anti-IFN-c (clone: XMG1.2) and anti-IL4 (clone: 11B11) were purchased from eBioscience, Frankfurt, Germany. Unconjugated anti-IL-2 (clone JES6-1A12) were obtained from Biolegend, San Diego, CA. Recombinant murine cytokines TNF-a, IL-1b, GM-CSF and IL-23 were all purchased from R&D systems, Wiesbaden-Nordenstadt, Germany whereas recombinant murine IL-6 was bought from Peprotech, Hamburg, Germany. 2.3. Preparation of primary mouse mixed glial cells Primary cultures of mixed glial cells were prepared from brains of postnatal 1–3 day old C57BL/6 mice. Briefly, the brains were freed from meninges, and digested enzymatically with 0.1% trypsin (Sigma–Aldrich) and 0.25% DNAse (Roche, Mannheim, Germany). Single cell suspensions obtained from the digested brains were seeded into poly-L-lysine-coated T 75 mm2 culture flasks in medium consisting of DMEM + L-Glutamine + 4.5 g/L D-Glucose (GibcoR, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS), 50 U/ml penicillin, and 50 lg/ml streptomycin (all Biochrom AG, Berlin, Germany). After 24 h all media containing cell debris was removed and fresh media was added. Medium was changed every fourth day and the microglia were harvested at day 9–11 by shaking the flask at 37 °C and 180 rpm for 30 min on an orbital shaker. 2.4. In vitro differentiation of Th1 and Th17 cells Naive CD4+ (CD25 CD62Lhi) from spleen and lymph nodes of C57BL/6J mice were sort purified using autoMACSPro (Miltenyi Biotec Bergisch Gladbach Germany). Cells (5.0  105/ml) were stimulated with plate-bound anti-CD3 (2 lg/ml) and anti-CD28 (2 lg/ml) in 12-well plates (Corning Life Science, Acton, MA) in complete IMDM (IMDM, 10% FCS, 1 mM Sodiumpyruvate 50 uM b-mercaptoethanol, 25 mM HEPES and non-essential amino acids) supplemented with either Th1-polarizing factors IL-12 (20 ng/ml), anti-IL4 (10 ug/ml) or with Th17-polarizing factors TGF-b1 (2 ng/ ml), IL-6 (30 ng/ml), TNF-a (20 ng/ml), IL-1b (10 ng/ml), anti-IL-2 (10 lg/ml), and anti-IFN-c (10 lg/ml). After five and six days culture of Th1 and Th17 cells, respectively, the cells were harvested at the end of the culture period, washed extensively and restimulated in 12-well plates coated with anti-CD3 and anti-CD28 antibodies for 6 h. In some experiments the restimulation was carried out in the presence of mouse recombinant IL-23 (20 ng/ ml). Supernatants were collected and stored at 80 °C until further use. For determining the purity of Th1 and Th17 cultures, cells were harvested and restimulated in the presence of Phorbol 12myristate 13-acetate (PMA; 10 ng/ml; Sigma) and ionomycin (500 ng/ml) before staining for intracellular cytokines. 2.5. Intracellular cytokine staining In vitro differentiated Th1 and Th17 cells were restimulated in 96-well plates in the presence of PMA (10 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma–Aldrich) and Brefeldin A (5 lg/ml; Sigma– Aldrich). The surface markers were stained with flurochrome conjugated anti-CD3 and anti-CD4 antibodies. To eliminate dead cells from the analysis Live/Dead staining was performed using Live/Dead fixable blue dead cell stain kit. Cells were washed thoroughly, fixed and permeabilized using the Foxp3 staining buffer set (eBioscience, Frankfurt, Germany) according to the manufacturer’s recommendations. Appropriate dilutions of antibodies for intracellular cytokines anti-IFN-c and anti-IL-17A were added and incubated for 30 min. Cells were washed and recorded immediately

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on an LSRII or Fortessa (BD Biosciences, Heidelberg, Germany). The data was analyzed by using the FlowJo software (Tree Star, USA). 2.6. Stimulation of microglia with Th1- and Th17-derived supernatants After harvesting, 2–5  105 microglia were plated in 6-well or 12-well plates and incubated for 24 h to regain their resting phenotype. After aspirating the existing medium, 1 ml of Th1- or Th17-derived culture supernatants diluted with equal volume of DMEM containing 10% FCS was added to the plated microglia. In some experiments, microglia were treated with Th17-derived supernatants supplemented with recombinant mouse GM-CSF (5 ng/ml). For flow cytometry the cells were released by adding pre-warmed 1 trypsin-EDTA solution. After blocking the Fcreceptors with anti-CD16/32, cells were stained with fluorochrome conjugated anti-mouse anti-CD11b, anti-CD40, anti-I-A/I-E and anti-CD86 antibodies. Rat anti-mouse IgG2a j and rat anti-mouse IgG2b j were used to stain the isotype controls. The cells were immediately analyzed on FACScalibur™ (BD Biosciences Heidelberg, Germany). 2.7. Co-culture of microglia and T cells Microglia harvested from the mixed glial cultures were plated in 12-well plates at 2.5  105 cells/well. Polarized Th1 or Th17 cells were restimulated with plate bound anti-CD3 and anti-CD28 antibodies for 6 h and added to microglia at 5  105 cells/well. After 16 h of co-culture the T cells were collected separately by gently pipetting the medium and microglia were harvested for flow cytometry by adding pre-warmed 1 trypsin EDTA. 2.8. Nitrite measurement NO production was measured indirectly by measuring the stable end product nitrite in the cell culture media. An aliquot of the cell culture media (100 ll) was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 min. The absorbance was measured at 490 nm. The concentration was calculated by linear regression to a standard curve. 2.9. MCP-1/CCL-2 ELISA Supernatants of microglia cultured under different conditions were collected from several experiments and were stored in aliquots at 80 °C until further use. CCL-2 in culture supernatants was measured using an enzyme-linked immunosorbent assay (ELISA) for mouse MCP-1/CCL-2 (R&D Systems), and carried out according to the manufacturer’s instructions. A standard curve was generated in the concentration range 0–250 pg/ml using the MCP-1 standard provided in the kit. The standard curve was calculated by a computer-generated four-parameter log (4-PL) fit curve. 2.10. Gene expression analysis Microglia were harvested from the 6-well plates by mild trypsinization and washed thoroughly in PBS. RNA was isolated from the cell pellet using the RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions. Quality and integrity of the total RNA was controlled on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). 500 ng of total RNA were applied for Cy3-labelling reaction using the one color Quick Amp Labeling protocol (Agilent Technologies; Waldbronn, Germany). Labeled cRNA was hybridized to Agilent´s murine 4  44 k microarrays for 16 h at 68 °C and scanned using the Agilent DNA Microarray Scanner. Expression signal values were

calculated by the software package Feature Extraction 10.5.1.1 (Agilent Technologies; Waldbronn, Germany). For further statistical analysis raw intensities were log 2 transformed and expressed as mean centralized relative gene intensity. Statistical analysis of the expression data was performed using the Gene Spring Software package (Agilent Technologies; Waldbronn, Germany). A background subtraction method eliminates all the genes whose detection flag was negative in any of the three conditions. Statistical T-Test were applied to identify differentially expressed gene sets using a p-value less than 0.05 and showing a fold change of more than two-fold. The entire microarray data set is available under Gene Expression Omnibus (GEO) accession number: GSE45384 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE45384). 2.11. Th1/ Th17 cytokine profiling (Luminex assay) Cytokine profiling was done using the mouse Th17 magnetic bead panel (Millipore) designed to simultaneously detect Th1 and Th17 cytokines (GM-CSF, TNF-a, IFN-c, IL-1b, IL-2, IL-10, IL17A, IL-17F, IL-21 and IL-22). Aliquots of cell culture supernatants were analysed according to the manufacturer’s instructions. Briefly, after removing cellular debris by centrifugation, 25 ll supernatant was mixed with 25 ll magnetic beads covered with catch antibodies against the cytokines listed above. After overnight incubation at 4 °C and washing, the magnetic beads were resuspended in 25 ll detection antibody solution. After one-hour incubation at RT 25 ll Streptavidin–Phycoerythrin was added to each sample and incubated for 30 min at RT. Finally, the beads were washed twice and resuspended in 150 ll sheath fluid. Beads incubated with standard proteins, control proteins and supernatants were measured on a MAGPIX (Millipore, Billerica, MA) and analyzed with the xPONENT 4.2 software (Luminex, Austin, Texas). The determination of the cytokine concentration was based on the standard curve (5- or 4-parameter logistic method). 2.12. CNS leukocyte isolation and flow cytometry CNS microglia were isolated from whole brain homogenates as described previously (de Haas et al., 2008) with modifications. In brief, mice were perfused transcardially with ice cold PBS until flow through was completely clear to remove intravascular leukocytes. After dissection brains were grinded in Hank’s Balanced Salt Solution (HBSS, Gibco, Eggenstein) using a tissue homogenizer (glass Potter, Braun, Melsungen) followed by a needle (0.6  25) and a syringe (5 ml) before passing through a 70 lm cell strainer (BD biosciences, Heidelberg). After pelleting, homogenates were resuspended in 75% isotonic Percoll (GE-healthcare, Uppsala, Sweden) at 4 °C. A discontinuous Percoll density gradient was layered as follows: 75%, 25% and 0% isotonic Percoll. The gradient was centrifuged for 25 min, 800g at 4 °C. Microglia, leukocytes, and astrocytes were collected from the 25%/75% interface. For surface marker staining the collected cells were directly washed in PBS, and blocked with CD16/CD32 (Fc block; eBioscience, Frankfurt/ Main, Germany) antibody. Isolated leukocytes were incubated with fluorochrome-conjugated antibodies (eBioscience) to detect CD11b (APC), CD45 (FITC), CD45 (eFluor 450), I-A/I-E. After washing, bound Ab was detected using a BD FACSCanto II (BD Biosciences), and the acquired data were analyzed using the flow cytometry software, FlowJo (TreeStar, San Carlos, CA). 2.13. Statistical analysis All statistical analyses were conducted using GraphPad Prism 5.0 (GraphPad Software). All data are expressed as group mean ± SD unless otherwise stated. All experiments were performed multiple times and the data obtained was analyzed using

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one-way ANOVA with Tukey’s post test. Results were considered statistically significant at p < 0.05. 3. Results 3.1. In vitro differentiation and characterization of Th1 and Th17 cells In order to study the effectiveness of effector molecules secreted by Th1 and Th17 cells in activating and inducing a robust inflammatory response in microglia our initial efforts were to establish in vitro cultures that yielded high percentage of cells producing IFN-c and IL-17, respectively. Naïve CD4+ T cells were sorted from the spleen and lymph nodes of mice and were cultured under Th1- or Th17-polarizing conditions. After five (Th1) or six (Th17) days of culture, the cells were harvested, restimulated and their phenotype was assessed by intracellular staining for IFN-c and IL-17A. We consistently achieved >99% of IFN-c-producing Th1 cells after five days of culture, whereas the yield of Th17 cells was slightly variable ranging from 60% to 90% of IL-17A-producing cells at day 6 of the culture. Importantly, Th1 cells did hardly express IL-17A and Th17 cells were largely devoid of IFNc-producing cells (