Article ID: WMC001727 ISSN 2046-1690

Differential Functions of CCR9- or CXCR3-Expressing Microglia

Author(s):Dr. He Yuling, Dr. Li Li, Dr. Wei Xiao , Dr. Xinti Tan , Dr. Mengjun Wu , Dr. Jie Li , Dr. Lan Wang , Dr. Lang Chen , Dr. Ruijing Xiao , Mr. Wei Huang, Mr. Xiaoling Zheng , Dr. Rui Zhou , Dr. Xiang Ji , Dr. Jie Xiong, Dr. Yanping Jiang , Dr. Jinquan Tan Corresponding Author: Dr. Jinquan Tan, Professor, Immunology, Wuhan University School of Medicine - China Submitting Author: Dr. Li Li, Student, Immunology, Wuhan University School of Medical, 430071 - China

Article ID: WMC001727 Article Type: Research articles Submitted on:13-Mar-2011, 04:36:03 AM GMT Published on: 14-Mar-2011, 10:07:04 PM GMT Article URL: http://www.webmedcentral.com/article_view/1727 Subject Categories:INFECTIOUS DISEASES Keywords:Microglia, , CCR9, CXCR3, Neurotoxic, Neurosupportive How to cite the article:Yuling H , Li L , Xiao W , Tan X , Wu M , Li J , Wang L , Chen L , Xiao R , Huang , Zheng X , Zhou R , Ji X , Xiong , Jiang Y , Tan J . Differential Functions of CCR9- or CXCR3-Expressing Microglia . WebmedCentral INFECTIOUS DISEASES 2011;2(3):WMC001727 Source(s) of Funding: The project was supported by the grants from the National Natural Science Foundation of China (30730054, 30572119, 30670937, 30971279, 30901363), by the Hi-tech Research and Development Program of China from Ministry of Science and Technology (2007AA02Z120), by the Ministry of Education (20060486008, 20090141120011), Provincial Department of Science and Technology of Hubei (2007ABC010), by Provincial Department of Health of Hubei (JX4B14), by Innovation Program of Wuhan University for Young Scholars (WU3082007), by National Innovation Experiment Program for College Students (WU2007061), China, and by Chang Jiang Scholars Program from Ministry of Education, China and Li Ka Shing Foundation, Hong Kong, China (Chang Jiang Scholar T.J.).

Competing Interests: No

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Differential Functions of CCR9- or CXCR3-Expressing Microglia

Abstract number of mechanisms. One hand, microglia are promoting the regeneration of neurons, on the other hand, microglia are inducing apoptosis of neurons [2, 4]. These different functions are highly dependent Underlying mechanisms by which how microglia upon the particular conditions that provoke activation accomplishes a destructive or constructive role in of microglia [2]. In our previous study, we have central nervous system remain to be fully studied. We reported that intracranially challenging and have established different mouse models by re-challenging with T. gondii in mouse model induces intracranially infection and re-challeng with the neurodestructive CCR9+Irg1+ (immunoresponsive Toxoplasma gondii (T. gondii) or infection and gene 1) microglia that are resistant to apoptosis, re-challeng with lymphocytic choriomeningitis virus secret large amount of TNF-a; Challenging and (LCMV). The neurotoxic CCR9+Irg1+ re-challenging with lymphocytic choriomeningitis virus (immunoresponsive gene 1) microglia are high (LCMV) induces neurosupportive CXCR3+Irg1- frequent in the brain in the mice infected and microglia that are sensitive to apoptosis, secret large re-challenged with T. gondii. These cells possess amount of IL-10 and TGF-b, suggesting that there are resistance to apoptosis and TNF-a-biased. The two different types of microglia [5]. However, by what neurosupportive CXCR3+Irg1- microglia are high mechanisms determining microglia to accomplish frequent in the brain in the mice infected and destructive or supportive role in central nervous re-challenged with LCMV. These cells are sensitive to system remain to be fully studied. apoptosis and IL-10- and TGF-b-biased. Moreover, the brain-derived neurotrophic factors (BDNF) express in Unlike other organs, central nervous system is one similar pattern in different CCR9+Irg1+ or CXCR3+Irg1- that has an immunologically privilege since a relative microglia. CCL25/CCR9 induces Irg1 phosphorylation impermeable blood-brain barrier and an immune in neurotoxic CCR9+Irg1+ microglia ex vivo. These suppression condition limiting the entry and function of data plus our previous report indicate that there are immune cells. However, microglia may provide with a two different subsets of microglia, which have direct initial response to neurotropic pathogenesis [6]. neurotoxic or neurosupportive function. The study It is necessary that the closeness and membrane provide with a number of novel evidences on contact between microglia and neurons to facilitate involvement of microglia in neurodegenerative and cell-cell communication via different signal features – neuroinflammatory diseases. soluble and diffusible factors, and their ligands and receptors such as and corresponding Introduction receptors [7]. A series of chemokines/receptors are considered as related molecules of central nervous system development and potential mediators for the Microglia, as key mediating cells of neurodegenerative immune-related neurodegenerative and and neuroinflammatory pathogenesis, heavily and neuroinflammatory pathogeneses in central nervous actively participate into the innate immune system of system [8]. For example, CXCR3/CXCL10 ligation is central nervous system [1]. The immune defense vital important for microglia migration and recruitment, capacities of microglia are "normal" in normal central and a basic factor for the reorganization of neurons [7, nervous system. However, these microglia are able to 9]. For another example, CCR9/CCL25 ligation is also be rapidly activated upon the pathogenesis such as an important element for T cells in their homeostasis, various inflammations in nervous system, chronic development, homing, and resistance to apoptosis [10]. central nervous system diseases, or brain damage [2, In our previous study, we have described the CCR9+ 3]. It is believed that the activated microglia are the Irg1+ and CXCR3+Irg1- microglia in the brain in first cells to react to the damage of neurons in the different mouse models after different priming and central nervous system. These activated microglia re-challenging, which possess different possess two opposing functions by means of a neurosupportive and neurodestructive founctions,

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respectively [5]. However, functional expression of infiltrating CD3+ T cells and CD19+ B cells were CCR9 on microglia still needs to be fully studied. positively depleted from brain cell suspensions by The expression of the Irg1 is up-regulated in mouse using anti-CD3 and anti-CD19 mAb labeled magnetic macrophages by bacterial LPS [11]. This molecule is Dynabeads (Dynal, Norway). The microglia in functionally involved in neurosupportive microglia [5, cell-suspension were stained with anti-mouse CD11b 12] and embryo implantation [11, 14]. In humans, the (Mac-1) and CD45 [7, 9]. Cells in cell-suspension were Irg1 gene has been predicted by bioinformatics tools, then stained with appropriate secondary however the existence of this gene is unconfirmed, fluorescence-labeled antibodies. To detect CCR9, and its expression pattern and function are unknown. CXCR3 or Irg1, the cells was stained by goat anti-mouse CCR9 pAb (E-15, Santa Cruz Materiials and Methods Biotechnology, Santa Cruz, CA) or goat anti-mouse CXCR3 pAb (SC-6226, R&D Systems, Abingdon, UK), or rabbit anti-mouse Irg1 (generated in-house) for the Animal models third color, respectively. In some specific cases, CD11b-CD45-CD3-CCR9 or -CXCR3 in Unless otherwise indicated, we had established cell-suspension was applied to distinguish microglia following animal models of mice that were used in populations from T cells, and to detect expression of whole study procedural: C57BL/6 mice, aged 5- to CCR9 and CXCR3 on different microglia cell 6-wk, were purchased from The Jackson Laboratory. populations. An intracellular immunofluorence staining The mice were intracranially injected three times with procedure of IntraPrepâ (Coulter-ImmunoTech, Miami, 8 mg T. gondii (DX strain) soluble tachyzoite antigen FL) was applied to permeabilize cells in Irg1 staining. (STAg) (referred to as Tp) [15], or intracranially To detect apoptosis of microglia and neurons, the injected three times with 0.1 mg recombinant microglia were isolated using the high-gradient nucleoprotein immunodominant domain of LCMV magnetic CD11b/Mac1-labelling cell separation (rLCMVNP) (referred to as Lp) [16] in biweekly time system (MACS, Milteny Biotec, Beijing, China) [9]. The point of intervals; After 2 weeks post-challenging, Tp microglial apoptosis was examined using propidium mice were intracranially infected (re-challenged) with 5 iodide binding and annexin V [19]. The cultures of cysts of T. gondii once (sublethal, low-virulent DX neuronal were isolated from cerebral cortices of fetal strain, referred to as TpTi); Lp mice were intracranially mouse [20]. The co-cultures of neuron and microglia infected with 100 plaque-forming units (pfu) of LCMV were kept for 36 h [20]. After treatment, the microglia one time (sublethal, clone 13, referred to as LpLi) [17, in co-cultures of neuron and microglia were removed, 18]; In some specific cases, Tp mice were infected and the left neurons were stained by the terminal with 100 pfu of LCMV one time (referred to as TpLi); deoxynucleotidyl transferase-mediated dUTP In other specific cases, Lp mice were infected with 5 nick-end-labeling method (Pheonix Flow Systems, San cysts of low-virulent DX strain T. gondii (referred to as Diego, CA) [21], The results were acquired by a flow LpTi); Un-primed mice were intracranially infected with cytometer (Moflow, Coulter Corporation, Miami, 5 cysts of low-virulent DX strain of T. gondii (a single Florida, USA). dose, referred to as Ti) or with 100 pfu of LCMV (a single dose, referred to as Li). The all animal model Western blotting mice were feed in a pathogen-free environment in the To detect the immune-precipitations and Animal Research Institute, Medical Research Center, phosphorylated , the purified cells or the cells in Wuhan University School of Medicine (Wuhan, China). cell-suspension were extensively washed, and lysed in The projects were approved by the Ethic Committee of the lysis solution [22]. Cell lysates were centrifuged Wuhan University, Wuhan. (9,000 rpm for 15 min, 4°C). The cell-supernatants Flow cytometry were obtained. The cell-supernatants were pre-cleared for three times with 20 ml of protein A-Sepharose To analyze the frequency and functions of microglia, beads. The pre-cleared cell-supernatants were added the cortex or hippocampus in the brains were with specified Ab (anti-Irg1) for 180 min at 4°C under micro-dissected from different mouse model mice at constant agitation. To obtain immune-complexes, the different intervals of time at indicated. The single-cell cell-supernatant were mixed with 20 ml of protein suspensions of the brains were made by using a A-Sepharose beads to allow binding for overnight, 70-mm mesh, the samples were then digested with deposited beads were washed for three times with collagenase/DNAse (Sigma), and separated with lysis solution, The immune-precipitates were Percoll (Pharmacia) gradient. Avoiding contamination separated in 12% SDS gels, and blotted onto the of T- and B-cells during flow cytometry analysis,

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nitrocellulose membranes. The membranes were blocked with 5 % nonfat milk in block solution, and cultured with rabbit anti-phosphorylated T. gondii- or LCMV-infection induces distinct antibody (Zymed Laboratories Inc., San Francisco, CA) microglia for 120 min, then incubated with horseradish We established two major mouse models to induce peroxidase-labeled secondary antibody (Amersham activated microglia in vivo [5] in order to examine what Pharmacia) to visualize the bend of phosphorylated factor(s) to decide differential function of stimulated protein, and recorded by autoradiography. In Western microglia for regeneration or degeneration of neurons. blot assay, protein concentration in the We previously reported that distinctive microglia from cell-supernatant was determined by Bio-Rad protein different animal models different expressed CXCR3 assay [23]. The certain amount of protein (around 40 and CCR9 [5]. A high level of CXCR3 expression was mg) was added onto 16 % SDS-PAGE. After protein found on the microglia from Lp and LpLi mice [5]. electrophoresis, the proteins in the SDS-PAGE were CCR9 expression was found significantly increased on blotted onto PVDF membranes. The membranes were the microglia from the brain of mice infected with T. incubated with the corresponding Abs (CCR9, CXCR3, gondii (Ti mice), whereas, CXCR3 expression was the and Irg1) at 0.5-0.8 mg/ml. The blotted membrane was majority of the infiltrating microglia to react blocked in 5% BSA-TBS, and incubated with inflammation in cortex and hippocampus region from secondary antibody, followed with to visualize the LCMV-infected mice (Li mice) (data not shown). To bend of target protein, and recorded by distinguish microglia populations from T cells, a autoradiography [21]. four-color flow cytometry (combination of staining of Q-RT-PCR CD11b-CD45-CD3-CCR9 or -CXCR3) was applied in cell-suspension from different mouse models. For The total RNA was isolated from the samples. All real detection of CD11b+CD45+ microglia populations, time quantitative RT-PCR (Q-RT-PCR) reactions were microglia from the brain of Tp and TpTi mice contacted out as what described previously [24]. In significantly and highly expressed CC chemokine brief, total RNA was isolated from target cells or brain receptor CCR9, whereas, the microglia from the brain tissues using a combination of Trizol (Invitrogen) and highly expressed CXC CXCR3 in the RNeasy mini (Qiagen), and reverse-transcribed Lp and LpLi mice (Fig. 1A), compared with normal TM with Oligo (dT) primer and using RevertAid First + + 12-18 mice. Meanwhile, for detection of CD3 CD45 Strand cDNA Synthesis kit (Fermentas). The DNAs in populations, the infiltrating CD3+ T cells expressed the tissues from T. gondii-infected animals tissues both CCR9 and CXCR3 at very low levels (Fig. 1B). were extracted using the Qiamp tissue kit (QIAGEN). These data strongly confirmed that we observed that Q-PCRs were performed with an ABI PRISMâ 7300 distinctive up-regulation of expression of CCR9 and Sequence Detector Systems (Applied Biosystems) by CXCR3 was indeed on activated microglia, but not on â using SYBR Green PCR Core Reagents Kit (Applied contaminating infiltrating T cells. Biosystems) according to the manufacturer¢s instructions. Distinctive functions of microglia from different animal models ELISA Analysis A very low frequency of microglia in apoptosis was Breifly, the supernatant from microglia cell-culture found in both Tp and TpTi mice. Meanwhile, a very containing cytokines and chemokines were examined high frequency of microglia in apoptosis was found in in the conditioned media by using ELISA kits both Lp and LpLi mice, compared with those cells in (PromoCell GmbH) [25]. The supernatant from normal mice. By TUNEL assay, the frequency of microglia cell-culture was collected from target cell microglia in apoptosis in cortex and hippocampus (microglia) cultures after post-incubation for the were detected significantly higher level in Lp and LpLi different intervals of time as indicated. The mice than that in microglia in apoptosis in Tp and TpTi supernatant from microglia cell-culture were mice in vivo, strongly suggesting that microglia in Lp pre-cleared at 2500 rpm for 8 min, and then assessed and LpLi were highly more sensitive to apoptosis (Fig. for target proteins (cytokines and chemokines) 2, some data not shown). In co-culture of neuron and according to the manufacturer's instructions. The microglia, the very high frequency of apoptotic assays were contact out for 6 times in every group, neurons were found in cell culture from Tp and TpTi and every sample was assessed in triplicate mice, whereas, very low frequency of apoptotic Results neurons in cell culture from Lp and LpLi mice, compared with these cell culture in NML mice [5].

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Furthermore, the microglia from Lp mice significantly The different cortical microglia expressed same inhibited the effect of apoptotic induction of the pattern of level of BDNF as hippocampal microglia microglia from Tp mice on neurons in vitro (Fig. 3). We (data not shown). further observed that the similar levels of IL-2, IFN-g CCL25/CCR9 induces Irg1 phosphorylation in and IL-4 were synthesized and secreted at mRNA and neurotoxic microglia protein levels in cell culture system of microglia from Irg1, a novel immune-related gene, intracellularly different mouse models including Tp, TpTi, Lp, and expressed a very high in CCR9-expressing Tp and LpLi mice (Fig. 4, and some data not shown). TNF-a in TpTi cortex microglia from mouse models, but was not Tp and TpTi microglia in the brain from mouse models found in the CXCR3-expressing microglia from NML, express significantly higher amount than that in the Lp and LpLi mouse models [5], and that the amount of microglia from Lp and LpLi mouse models, whereas, phosphorylated-Irg1 proteins were dependent on IL-10 and TGF-b in Lp and LpLi microglia in the brain ligation of CCR9/CCL25 [5]. In the present study, we from mouse models were synthesized significant further demonstrated that ligation of CCR9 and CCL25 higher levels than that in the cells from Tp and TpTi was able to directly initiate Irg1 phosphorylation in the mouse models (Fig. 4, and some data not shown). We freshly isolated CCR9+Irg1+ Tp and TpTi microglia have examined the expression levels both at mRNA from mouse models in comparison with NML microglia and protein levels (Fig. 4, and some data not shown). (Fig. 7). CCL25 in Tp and TpTi microglia the brain from mouse models were synthesized significant higher level than Discussion the Lp and LpLi microglia from mouse models. CX3 CL1 and CXCL10 in Lp and LpLi microglia from mouse models were synthesized significant higher level than Microglia are believed as the major cellular part of the these Tp and TpTi microglia from mouse models at brain immune system in the central nervous system [1]. mRNA level at mRNA and protein levels (Fig. 4, and In the normal brains, the infiltrations of eukocytes are some data not shown). The similar levels of CCL5 and lack the prominent in adaptive immunities. However, in CXCL12 at mRNA and protein levels were synthesized the neuroinflammation and neurodegeneration in these microglia from either Tp and TpTi or Lp and conditions, activated microglia are infiltrating to initiate LpLi mouse models (Fig. 4, and data not shown). The the different pathogeneses [1, 4, 26]. Therefore, Tp and TpTi microglia from mouse models secreted activated microglia-related innate immunity in the three to fivefold more nitrite than that NML, Lp and central nervous system is thought to be a possible LpLi microglia from in mouse models [5]. We further pathogenic component in many brain diseases [1, 4, analyzed the inhibitory effect of nitric oxide inhibitor on 26]. The unanswered questions are if activation of toxicity of neurotoxic microglia toward neurons. microglia in vivo is able to be directly initiated by Different microglia were incubated without or with nitric pathophysiological molecules, which are not derived oxide inhibitor Nw-nitro-l-arginine methyl ester (l-NAME) and secreted from neuron, and if the activation of prior to apoptosis assay. The data revealed that NO microglia is capable of resulting in neurotoxicity of inhibitor l-NAME could significantly inhibit apoptotic microglia towards normal neurons [4]. Resolving these effect of microglia from Tp and TpTi mouse models on questions is particularly important for understanding of neurons in vitro (Fig. 5), indicating that apoptotic effect neurodegenerative and neuroinflammatory diseases of Tp and TpTi microglia was by means of increased such as Alzheimer's disease and AIDS dementia. In production of NO. To find more direct evidence on these pathogeneses, the chronic central nervous neurotoxic and neurosupportive effects of different system inflammatory process is considered to play a CXCR3+Irg1- and CCR9+Irg1+ microglia in vivo, we crucial role [26]. In our previous study, we have examined the expression of a member of the established two distinctive mouse models by either neurotrophin family (NGF, BDNF, NT3, and NT4), intracranial infecting and re-infecting with T. gondii, in brain-derived neurotrophic factors (BDNF), in different which induces the neurodestructive, microglia. The data by real time quantitative RT-PCR apoptosis-resistant and TNF-a-biased CCR9+Irg1+ assays revealed that mRNA BDNF in normal microglia microglia, or by infecting and re-infecting with LCMV, from hippocampus were at low level, and not changed in which induces neurosupportive, apoptosis-sensitive in Tp, TpTi, Lp and LpLi mouse models. Meanwhile, and IL-10- and TGF-b-biased CXCR3+Irg1- microglia, mRNA expression of BDNF was at relative high level strongly suggesting that two different types of in hippocampus tissues from NML mice, and was even microglia with different functions are existed [5]. In the significantly up-regulated in Tp and TpTi mouse present study, in the two different mouse models, we models, but not in Lp and LpLi mouse models (Fig. 6). have further confirmed up-regulation of CCR9- and

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CXCR3-expressing microglia in different types of in pathogenesis of neuronal degeneration or mouse models, and rule out the possibility on regeneration in central nervous system. Particularly, infiltrating T cells in the inflammatory brain from the two distinctive subsets of the CXCR3+Irg1- and CCR9+ two mouse models (Fig. 1). Irg1+ microglia have distinctive secreting profiles in In the previous study, we have detected a related low cytokines and chemokines as well as expressions of frequency normal microglia in mice. Most of normal chemokine receptor in respect to neuronal killer and microglia from the un-challenged mice are CXCR3- neuronal supporter. In the present study, we have CCR9-Irg1-, and functionally inactive in terms of their provided with more direct evidence to document the + - productions of cytokines and chemokines in vitro. effects of neurosupportive CXCR3 Irg1 and neurotoxic + + However, distinctive neurotoxic and neurosupportive CCR9 Irg1 microglia in Lp and LpLi mouse models as effects of Lp and LpLi CXCR3+Irg1- microglia and Tp well as Tp and TpTi mouse models in vivo. Moreover, and TpTi CCR9+Irg1+ microglia from different mouse we have also observed that ligation of CCR9 by models have been demonstrated in vitro in the CCL25 can directly cause phosphorylation of Irg1 ex + + previous study [5]. By TUNEL assay, the frequency of vivo in the freshly isolate Tp and TpTi CCR9 Irg1 Lp and LpLi microglia in apoptosis in cortex in mouse microglia from mouse models (Fig. 7). However, how models were significantly higher than that frequency of CCR9/CCL25/Irg1 pathway contributes to Tp and TpTi microglia in apoptosis in mouse models microglia-involved regeneration and death of neurons detected, strongly suggesting that microglia from Lp in vivo should be further investigated. and LpLi mice were more sensitive to apoptosis in vivo Acknowledgement(s) (Fig. 2), further providing with the functions of CCR9+ Irg1+ and CXCR3+Irg1- microglia, e.g. the neurotoxic, apoptotic resistant and TNF-a-biased CCR9+Irg1+ The project was supported by the grants from the microglia and the neurosupportive, apoptotic sensitive, National Natural Science Foundation of China and IL-10- and TGF-b-biased CXCR3+Irg1- microglia. (30730054, 30572119, 30670937, 30971279, However, we still need more direct evidences to show 30901363), by the Hi-tech Research and Development the functions of neurosupportive CXCR3+Irg1- and Program of China from Ministry of Science and neurotoxic CCR9+Irg1+ microglia in vivo. Moreover, we Technology (2007AA02Z120), by the Ministry of still need to collect more evidences on how CCR9+Irg1+ Education (20060486008, 20090141120011), and CXCR3+Irg1- microglia function as a neuron killer Provincial Department of Science and Technology of and supporter in vivo. Hubei (2007ABC010), by Provincial Department of Many intrinsic factors in the neurons of central nervous Health of Hubei (JX4B14), by Innovation Program of system control and elaborate the immune functions of Wuhan University for Young Scholars (WU3082007), activated microglia. The membrane-bound and by National Innovation Experiment Program for secreted factors from neurons and microglia, such as College Students (WU2007061), China, and by Chang cytokines and chemokines, can control the immune Jiang Scholars Program from Ministry of Education, activity in the central nervous system [7, 9, 27 - 30]. China and Li Ka Shing Foundation, Hong Kong, China For example, the cytokines and chemokines play an (Chang Jiang Scholar T.J.). important role of the communication and effector functions of microglia [28, 31]. The expression levels References of TNFa, IL-6, and IFN-g in microglia have critically involved in both protective and harmful neurons in central nervous system. The expression of CCR9 is 1. Medzhitov, R., and C. Janeway Jr. 2000. Advances up-regulated during T-cell development [32, 33]. A in immunology: innate immunity. N. Engl. J. Med. 343: considerable number of investigations has shown that 338–344. pair of CCR9/CCL25 is an important role for the T cell 2. Streit, W. J., S. A. Walter, and N. A. Pennell. 1999. development, homing, homeostasis, particularly, Reactive microgliosis. Prog. Neurobiol. 57: 563–581. mucosal T cells [34, 35]. In our previous study, 3. Fischer, H. G., and G. Reichmann. 2001. Brain CCR9-expressing microglia is majority of cell dendritic cells and macrophages/microglia in central population of the Tp and TpTi microglia in mouse nervous system inflammation. J. Immunol. 166: models, and CXCR3-expressing microglia is majority 2717–2726. population of Lp and LpLi microglia in mouse models 4. Kreutzberg, G. W. 1996. Microglia: a sensor for [5]. Two distinctive subsets of the microglia from the pathological events in the CNS. Trends. Neurosci. 19: two different mouse models contribute distinctive roles 312–318.

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Illustrations

Illustration 1

Figure 1. The expression of CXCR3 or CCR9 on microglia and T cells from different animal models. The flow cytometric analyses by four-color of CCR9 or CXCR3 on microglia (A) and CD3 T cells (B) from NML, Tp, TpTi, Lp, LpLi mouse models. The staining combination was CD11b-CD45-CD3-CCR9 (upper panels) or -CXCR3 (lower panels). The mice were sacrificed either at end of challenging procedure or at post-infection day 10. The single cell suspensions from cortexes of different mouse models were made and stained. Microglial populations were determined by the expression of CD11b/Mac1hi and CD45int double positive (A). T cell populations were determined the expression of CD3 and CD45hi double positive (B). Red curves, the staining for CCR9; Pink curves, the staining of CXCR3; Blue curves, the staining of CD3; Black curves, the staining of CD11b; Gray curves, the staining of isotype Ab controls. The numbers in the figure were showing the positive cell percentages. Data were showing the representatives of 4 mice per group.

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Illustration 2

Figure 2: Measurement of apoptosis of microglia by TUNEL assay. NML, Tp, TpTi, Lp, and LpLi mice were sacrificed either at end of challenging procedure or at post-infection day 10. The cortex samples were made into frozen cryosections (6 µm thick). Apoptotic nuclei were detected by using TUNEL technique (Molina-Holgado E. et al., 2002 J. Neurosci. 22:9742). TUNEL reactions were performed for 60 minutes at 37°C using In Situ Cell Death Detection Kit (Roche Diagnostics Corp.) according to the manufacturer’s instructions. The microglia were also stained with CD11b/Mac1 antibody. Using a fluorescence microscope, TUNEL-positive microglia were counted, and verified by CD11b/Mac1 staining. A mean value of per high power field (hpf) of 3 sections per animal was used for analysis. Data were represented as mean ± SD of 3 animals per group.

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Illustration 3

Figure 3. Interaction of killer and supportive microglia by analysis of flow cytometry of neurons in apoptosis in culture. Neurons were isolated from cerebral cortices of fetal mice (embryonic day 18), and co-cultured with microglia isolated from Tp and Lp mice with different ratio with LPS in culture for 48h, and added with pyrimethamine and ganciclovir as described in Materials and Methods. A, without microglia; B, NML microglia; C, Tp microglia; D, Lp microglia; E, Tp : Lp microglia = 1 : 1; F, Tp : Lp microglia = 1 : 5; G, Tp : Lp microglia = 5 : 1. The FITC-conjugated Brdu stained neurons were analyzed. The pictures for neurons in apoptosis were from representatives of 4 conducted experiments.

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Illustration 4

Figure 4. The production of cytokines and chemokines from different microglia from different mice (including NML, Tp, TpTi, Lp, LpLi mouse models). The cytokine levels (upper panels) and chemokine levels (lower panels) microglial culture supernatant, which are infiltrating into inflammatory cortex assessed by ELISA. Mice were sacrificed either end of challenging procedure or at post-infection day 10. The high-gradient magnetic CD11b/Mac1-labelling cell separation system was used to isolate the microglia (n = 4), and the isolated microglia were cultured with LPS in the system for 6h added with pyrimethamine and ganciclovir. In the upper leftmost panel, white bars, IL-2; black bars, IFN-?. *p < 0.01 - 0.001, NML, Lp, or LpLi versus Tp or TpTi for IL-10 and TGF-?;*p < 0.001, Tp or TpTi versus NML, Lp, or LpLi for TNF-?; *p < 0.001, Tp or TpTi versus NML, Lp, or LpLi for CCL25; *p < 0.001, Lp or LpLi versus NML, Tp or TpTi for CX3CL1 and CXCL10.

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Illustration 5

Figure 5. Analysis of inhibitory effect of nitric oxide inhibitor on toxicity of neurotoxic microglia toward neurons. Analysis of apoptotic neurons in culture was conducted by flow cytometry. Neurons were isolated from cerebral cortices of fetal mice (embryonic day 18), and co-cultured with microglia isolated from Tp and Lp mice with different ratio with LPS in culture for 48h, and added with pyrimethamine and ganciclovir as described in Materials and Methods. Different microglia were incubated without or with nitric oxide inhibitor N?-nitro-l-arginine methyl ester (l-NAME, 10 µm; Sigma) prior to apoptosis assay (Layton CJ, et al. J. Neurochem. 2005, 92:487). The l-NAME was present during the apoptotic culture period. A, NML microglia (without l-NAME-treatment); B, NML microglia; C, Tp microglia (without l-NAME-treatment); D, TpTi microglia (without l-NAME-treatment); E, Tp microglia; F, TpTi microglia. The FITC-conjugated Brdu stained neurons were analyzed. The pictures for neurons in apoptosis were from representatives of 4 conducted experiments.

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Illustration 6

Figure 6. BDNF expression in purified hippocampal microglia and hippocampal tissue from different mice (including NML, Tp, TpTi, Lp, LpLi mouse models). Mice were sacrificed either end of challenging procedure or at post-infection day 10. Hippocampus was microdissected from mice. The high-gradient magnetic CD11b/Mac1-labelling cell separation system was used to isolate the microglia. The real time quantitative RT-PCR assays were performed as described in Materials and Methods (n = 4). The sequences of the primer for BDNF mRNA detection were senses 5'-GAGGGCTCCTGCTTCTCAA-3' and antisenses 5'-GCCTTCATGCAACCGAAGT-3'

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Illustration 7

Figure 7. Irg1 expression and phosphorylation in microglia from cortex of NML, Tp, and TpTi mice. Mice were sacrificed either end of challenging procedure or at post-infection day 10. T The high-gradient magnetic CD11b/Mac1-labelling cell separation system was used to isolate the microglia. The purified microglia were cultured at absence or presence of exogenous CCL25 (100 ng/ml) for 1 h before cell lysis. The Irg1 was immune-precipitated from the cell lysates. The levels of phosphorylation of Irg1 were detected using a rabbit anti-phosphorylated proteins Ab (n = 4)

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