Differential Functions Of CCR9- Or CXCR3-Expressing Microglia

How to cite the article: Yuling H, Li L, Xiao W, Tan X, Wu M, Li J, et al. Differential Functions of CCR9- or CXCR3-Expressing Microglia. WebmedCentral INFECTIOUS DISEASES 2011;2(3):WMC001727
doi: 10.9754/journal.wmc.2011.001727

WebmedCentral Peer Reviewed: No

Submitted on: 13 Mar 2011 04:36:03 AM GMT

Published on: 14 Mar 2011 10:07:04 PM GMT

Abstract

Underlying mechanisms by which how microglia accomplishes a destructive or constructive role in central nervous system remain to be fully studied. We have established different mouse models by intracranially infection and re-challeng with Toxoplasma gondii (T. gondii) or infection and re-challeng with lymphocytic choriomeningitis virus (LCMV). The neurotoxic CCR9⁺Irg1⁺ (immunoresponsive gene 1) microglia are high frequent in the brain in the mice infected and re-challenged with T. gondii. These cells possess resistance to apoptosis and TNF-a-biased. The neurosupportive CXCR3⁺Irg1^- microglia are high frequent in the brain in the mice infected and re-challenged with LCMV. These cells are sensitive to apoptosis and IL-10- and TGF-b-biased. Moreover, the brain-derived neurotrophic factors (BDNF) express in similar pattern in different CCR9⁺Irg1⁺ or CXCR3⁺Irg1^- microglia. CCL25/CCR9 induces Irg1 phosphorylation in neurotoxic CCR9⁺Irg1⁺ microglia ex vivo. These data plus our previous report indicate that there are two different subsets of microglia, which have neurotoxic or neurosupportive function. The study provide with a number of novel evidences on involvement of microglia in neurodegenerative and neuroinflammatory diseases.

Introduction

Microglia, as key mediating cells of neurodegenerative and neuroinflammatory pathogenesis, heavily and actively participate into the innate immune system of central nervous system [1]. The immune defense capacities of microglia are "normal" in normal central nervous system. However, thesemicroglia are able to be rapidly activated upon the pathogenesis such as various inflammations in nervous system, chroniccentral nervous system diseases, or brain damage [2, 3]. It is believed that the activated microglia are the first cellsto react to the damage of neurons in the central nervous system. These activated microglia possess two opposing functions by means of a 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 upon the particular conditions that provoke activation of microglia [2]. In our previous study, we have reported that intracranially challenging and re-challenging with T. gondii in mouse model induces the neurodestructive CCR9⁺Irg1⁺ (immunoresponsive gene 1) microglia that are resistant to apoptosis, secret large amount of TNF-a; Challenging and re-challenging with lymphocytic choriomeningitis virus (LCMV) induces neurosupportive CXCR3⁺Irg1^- microglia that are sensitive to apoptosis, secret large amount of IL-10 and TGF-b, suggesting that there are two different types of microglia [5]. However, by what mechanisms determining microglia to accomplish destructive or supportive role in central nervous system remain to be fully studied.

Unlike other organs, central nervous system is one that has an immunologicallyprivilege since a relative impermeable blood-brainbarrier and an immune suppression condition limiting the entry and function of immune cells. However, microglia may provide with a directinitial response to neurotropic pathogenesis [6]. It is necessary that the closeness and membrane contact between microglia and neurons to facilitate cell-cell communication via different signal features – soluble and diffusible factors, and their ligands and receptors such as chemokines and corresponding receptors [7]. A series of chemokines/receptors are considered as related molecules of central nervous systemdevelopment and potential mediatorsfor the immune-related neurodegenerative and neuroinflammatory pathogenesesin central nervous system [8]. For example, CXCR3/CXCL10 ligation is vital important for microglia migration and recruitment, and a basic factor for the reorganization of neurons [7, 9]. For another example, CCR9/CCL25 ligation is also an important element for T cells in their homeostasis, development, homing, and resistance to apoptosis [10]. In our previous study, we have described the CCR9⁺Irg1⁺ and CXCR3⁺Irg1^- microglia in the brain in different mouse models after different priming and re-challenging, which possess different neurosupportive and neurodestructive founctions, respectively [5]. However, functional expression of CCR9 on microglia still needs to be fully studied.

The expression of the Irg1 is up-regulated in mouse macrophages by bacterial LPS [11].This molecule is functionally involved in neurosupportive microglia [5, 12]and embryo implantation [11, 14]. In humans, the Irg1 gene has been predicted by bioinformatics tools, however the existence of this gene is unconfirmed, and its expression pattern and function are unknown.

Materiials and Methods

Animal models

Unless otherwise indicated, we had established following animal models of mice that were used in whole study procedural: C57BL/6 mice, aged 5- to 6-wk, were purchased from The Jackson Laboratory. The mice were intracranially injected three times with 8 mg T. gondii (DX strain) soluble tachyzoite antigen (STAg) (referred to as Tp) [15], or intracranially injected three times with 0.1 mg recombinant nucleoprotein immunodominant domain of LCMV (rLCMVNP) (referred to as Lp) [16] in biweekly time point of intervals; After 2 weeks post-challenging, Tp mice were intracranially infected (re-challenged) with 5 cysts of T. gondii once (sublethal, low-virulent DX strain, referred to as TpTi); Lp mice were intracranially infected with 100 plaque-forming units (pfu) of LCMV one time (sublethal, clone 13, referred to as LpLi) [17, 18]; In some specific cases, Tp mice were infected with 100 pfu of LCMV one time (referred to as TpLi); In other specific cases, Lp mice were infected with 5 cysts of low-virulent DX strain T. gondii (referred to as LpTi); Un-primed mice were intracranially infected with 5 cysts of low-virulent DX strain of T. gondii (a single dose, referred to as Ti) or with 100 pfu of LCMV (a single dose, referred to as Li). The all animal model mice were feed in a pathogen-free environment in the Animal Research Institute, Medical Research Center, Wuhan University School of Medicine (Wuhan, China). The projects were approved by the Ethic Committee of Wuhan University, Wuhan.

Flow cytometry

To analyze the frequency and functions of microglia, the cortex or hippocampus in the brains were micro-dissected from different mouse model mice at different intervals of time at indicated. The single-cell suspensions of the brainswere made by using a 70-mm mesh, the samples were then digested with collagenase/DNAse (Sigma), and separated with Percoll (Pharmacia) gradient. Avoiding contamination of T- and B-cells during flow cytometry analysis, infiltrating CD3⁺ T cells and CD19⁺ B cells were positively depleted from brain cell suspensions by using anti-CD3 and anti-CD19 mAb labeled magnetic Dynabeads (Dynal, Norway). The microglia in cell-suspension were stained with anti-mouse CD11b (Mac-1) and CD45 [7, 9]. Cells in cell-suspension were then stained with appropriate secondary fluorescence-labeled antibodies. To detect CCR9, CXCR3 or Irg1, the cells was stained by goat anti-mouse CCR9 pAb (E-15, Santa Cruz 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 third color, respectively. In some specific cases, CD11b-CD45-CD3-CCR9 or -CXCR3 in cell-suspension was applied to distinguish microglia populations from T cells, and to detect expression of CCR9 and CXCR3 on different microglia cell populations. An intracellular immunofluorence staining procedure of IntraPrepâ (Coulter-ImmunoTech, Miami, FL) was applied to permeabilize cells in Irg1 staining. To detect apoptosis of microglia and neurons, the microglia were isolated using the high-gradient magnetic CD11b/Mac1-labelling cell separation system (MACS, Milteny Biotec, Beijing, China) [9]. The microglial apoptosis was examined using propidium iodide binding and annexin V [19]. The cultures of neuronal were isolated from cerebral cortices of fetal mouse [20]. The co-cultures of neuron and microglia were kept for 36 h [20]. After treatment, the microglia in co-cultures of neuron and microglia were removed, and the left neurons were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling method (Pheonix Flow Systems, San Diego, CA) [21], The results were acquired by a flow cytometer (Moflow, Coulter Corporation, Miami, Florida, USA).

Western blotting

To detect the immune-precipitations and phosphorylated protein, the purified cells or the cells in cell-suspension were extensively washed, and lysed in the lysis solution [22]. Cell lysates were centrifuged (9,000 rpm for 15 min, 4°C). The cell-supernatants were obtained. The cell-supernatantswere pre-cleared for three times with 20 mlof protein A-Sepharose beads. The pre-cleared cell-supernatants were added with specified Ab (anti-Irg1)for 180 min at 4°C under constant agitation. To obtain immune-complexes, the cell-supernatant were mixed with 20 ml of protein A-Sepharose beads to allow binding for overnight, deposited beads were washed for three times with lysis solution, The immune-precipitates were separated in12% SDS gels, and blotted onto the nitrocellulose membranes. The membranes were blocked with 5 % nonfatmilk in block solution, and cultured with rabbit anti-phosphorylated proteins antibody (Zymed Laboratories Inc., San Francisco, CA) for 120 min, then incubated with horseradish peroxidase-labeled secondary antibody (Amersham Pharmacia) to visualize the bend of phosphorylated protein, and recorded by autoradiography. In Westernblot assay, protein concentration in the cell-supernatant was determined by Bio-Rad protein assay [23]. The certain amount of protein (around 40 mg) was added onto 16 % SDS-PAGE. After protein electrophoresis, the proteins in the SDS-PAGE were blotted onto PVDF membranes. The membranes were incubated with the corresponding Abs (CCR9, CXCR3, and Irg1) at 0.5-0.8 mg/ml. The blotted membrane was blocked in 5% BSA-TBS, and incubated with secondary antibody, followed with to visualize the bend of target protein, and recorded by autoradiography [21].

Q-RT-PCR

The total RNA was isolated from the samples. All real time quantitative RT-PCR (Q-RT-PCR) reactions were contacted out as what described previously [24]. In brief, total RNA was isolated from target cells or brain tissues using a combination of Trizol (Invitrogen) and the RNeasy mini kit (Qiagen), and reverse-transcribed with Oligo (dT)_12-₁₈ primer and using RevertAid^TM First Strand cDNA Synthesis kit (Fermentas). The DNAs in the tissues from T. gondii-infected animals tissues were extracted using the Qiamp tissue kit (QIAGEN). Q-PCRs were performed with an ABI PRISMâ 7300 Sequence Detector Systems (Applied Biosystems) by using SYBR^â Green PCR Core Reagents Kit (Applied Biosystems) according to the manufacturer¢s instructions.

ELISA Analysis

Breifly, the supernatant from microglia cell-culture containing cytokines and chemokines were examined in the conditioned media by using ELISA kits (PromoCell GmbH) [25]. The supernatant from microglia cell-culture was collected from target cell (microglia) cultures after post-incubation for the different intervals of time as indicated. The supernatant from microglia cell-culture were pre-cleared at 2500 rpm for 8 min, and then assessed for target proteins (cytokines and chemokines) accordingto the manufacturer's instructions. The assays were contact out for 6 times in every group, and every sample was assessed in triplicate

Results

T. gondii- or LCMV-infection induces distinct microglia

We established two major mouse models to induce activated microglia in vivo [5] in order to examine what factor(s) to decide differential function of stimulated microglia for regeneration or degeneration of neurons. We previously reported that distinctive microglia from different animal models different expressed CXCR3 and CCR9 [5]. A high level of CXCR3 expression was found on the microglia from Lp and LpLi mice [5]. CCR9 expression was found significantly increased on the microglia from the brain of mice infected with T. gondii (Ti mice), whereas, CXCR3 expression was the majority of the infiltrating microglia to react inflammation in cortex and hippocampus region from LCMV-infected mice (Li mice) (data not shown). To distinguish microglia populations from T cells, a four-color flow cytometry (combination of staining of CD11b-CD45-CD3-CCR9 or -CXCR3) was applied in cell-suspension from different mouse models. For detection of CD11b⁺CD45⁺ microglia populations, microglia from the brain of Tp and TpTi mice significantly and highly expressed CC chemokine receptor CCR9, whereas, the microglia from the brain highly expressed CXC chemokine receptor CXCR3 in Lp and LpLi mice (Fig. 1A), compared with normal mice. Meanwhile, for detection of CD3⁺CD45⁺ T cell populations, the infiltrating CD3⁺ T cells expressed both CCR9 and CXCR3 at very low levels (Fig. 1B). These data strongly confirmed that we observed that distinctive up-regulation of expression of CCR9 and CXCR3 was indeed on activated microglia, but not on contaminating infiltrating T cells.

Distinctive functions of microglia from different animal models

A very low frequency of microglia in apoptosis was found in both Tp and TpTi mice. Meanwhile, a very high frequency of microglia in apoptosis was found in both Lp and LpLi mice, compared with those cells in normal mice. By TUNEL assay, the frequency of microglia in apoptosis in cortex and hippocampus were detected significantly higher level in Lp and LpLi mice than that in microglia in apoptosis in Tp and TpTi mice in vivo, strongly suggesting that microglia in Lp and LpLi were highly more sensitive to apoptosis (Fig. 2, some data not shown). In co-culture of neuron and microglia, the very high frequency of apoptotic neurons were found in cell culture from Tp and TpTi mice, whereas, very low frequency of apoptotic neurons in cell culture from Lp and LpLi mice, compared with these cell culture in NML mice [5]. Furthermore, the microglia from Lp mice significantly inhibited the effect of apoptotic induction of the microglia from Tp mice on neurons in vitro (Fig. 3). We further observed that the similar levels of IL-2, IFN-g and IL-4 were synthesized and secreted at mRNA and protein levels in cell culture system of microglia from different mouse models including Tp, TpTi, Lp, and LpLi mice (Fig. 4, and some data not shown). TNF-a in Tp and TpTi microglia in the brain from mouse models express significantly higher amount than that in the microglia from Lp and LpLi mouse models, whereas, IL-10 and TGF-b in Lp and LpLi microglia in the brain from mouse models were synthesized significant higher levels than that in the cells from Tp and TpTi mouse models (Fig. 4, and some data not shown). We have examined the expression levels both at mRNA and protein levels (Fig. 4, and some data not shown). CCL25 in Tp and TpTi microglia the brain from mouse models were synthesized significant higher level than the Lp and LpLi microglia from mouse models. CX₃CL1 and CXCL10 in Lp and LpLi microglia from mouse models were synthesized significant higher level than these Tp and TpTi microglia from mouse models at mRNA level at mRNA and protein levels (Fig. 4, and some data not shown). The similar levels of CCL5 and CXCL12 at mRNA and protein levels were synthesized in these microglia from either Tp and TpTi or Lp and LpLi mouse models (Fig. 4, and data not shown). The Tp and TpTi microglia from mouse models secreted three to fivefold more nitrite than that NML, Lp and LpLi microglia from in mouse models [5]. We further analyzed the inhibitory effect of nitric oxide inhibitor on toxicity of neurotoxic microglia toward neurons. Different microglia were incubated without or with nitric oxide inhibitor N^w-nitro-l-arginine methyl ester (l-NAME) prior to apoptosis assay. The data revealed that NO inhibitor l-NAME could significantly inhibit apoptotic effect of microglia from Tp and TpTi mouse models on neurons in vitro (Fig. 5), indicating that apoptotic effect of Tp and TpTi microglia was by means of increased production of NO. To find more direct evidence on neurotoxic and neurosupportive effects of different CXCR3⁺Irg1^- and CCR9⁺Irg1⁺ microglia in vivo, we examined the expression of a member of the neurotrophin family (NGF, BDNF, NT3, and NT4), brain-derived neurotrophic factors (BDNF), in different microglia. The data by real time quantitative RT-PCR assays revealed that mRNA BDNF in normal microglia from hippocampus were at low level, and not changed in Tp, TpTi, Lp and LpLi mouse models. Meanwhile, mRNA expression of BDNF was at relative high level in hippocampus tissues from NML mice, and was even significantly up-regulated in Tp and TpTi mouse models, but not in Lp and LpLi mouse models (Fig. 6). The different cortical microglia expressed same pattern of level of BDNF as hippocampal microglia (data not shown).

CCL25/CCR9 induces Irg1 phosphorylation in neurotoxic microglia

Irg1, a novel immune-related gene, intracellularly expressed a very high in CCR9-expressing Tp and TpTi cortex microglia from mouse models, but was not found in the CXCR3-expressing microglia from NML, Lp and LpLi mouse models [5], and that the amount of phosphorylated-Irg1 proteins were dependent on ligation of CCR9/CCL25 [5]. In the present study, we further demonstrated that ligation of CCR9 and CCL25 was able to directly initiate Irg1 phosphorylation in the freshly isolated CCR9⁺Irg1⁺ Tp and TpTi microglia from mouse models in comparison with NML microglia (Fig. 7).

Discussion

Microglia are believed as the major cellular part of the brain immune system in the central nervous system [1]. In the normal brains, the infiltrations of eukocytes are lack the prominent in adaptive immunities. However, in the neuroinflammation and neurodegeneration conditions, activated microglia are infiltrating to initiate the different pathogeneses [1, 4, 26]. Therefore, activated microglia-related innate immunity in the central nervous system is thought to be a possible pathogenic component in many brain diseases [1, 4, 26]. The unanswered questions are if activation of microglia in vivo is able to be directly initiated by pathophysiological molecules, which are not derived and secreted from neuron, and if the activation of microglia is capable of resulting in neurotoxicity of microglia towards normal neurons [4]. Resolving these questions is particularly important for understanding of neurodegenerative and neuroinflammatory diseases such as Alzheimer's disease and AIDS dementia. In these pathogeneses, the chronic central nervous system inflammatory process is considered to play a crucial role [26]. In our previous study, we have established two distinctive mouse models by either intracranial infecting and re-infecting with T. gondii, in which induces the neurodestructive, apoptosis-resistant and TNF-a-biased CCR9⁺Irg1⁺ microglia, or by infecting and re-infecting with LCMV, in which induces neurosupportive, apoptosis-sensitive and IL-10- and TGF-b-biased CXCR3⁺Irg1^- microglia, strongly suggesting that two different types of microglia with different functions are existed [5]. In the present study, in the two different mouse models, we have further confirmed up-regulation of CCR9- and CXCR3-expressing microglia in different types of mouse models, and rule out the possibility on infiltrating T cells in the inflammatory brain from the two mouse models (Fig. 1).

In the previous study, we have detected a related low frequency normal microglia in mice. Most of normal microglia from the un-challenged mice are CXCR3^-CCR9^-Irg1^-, and functionally inactive in terms of their productions of cytokines and chemokines in vitro. However, distinctive neurotoxic and neurosupportive effects of Lp and LpLi CXCR3⁺Irg1^- microglia and Tp and TpTi CCR9⁺Irg1⁺ microglia from different mouse models have been demonstrated in vitro in the previous study [5]. By TUNEL assay, the frequency of Lp and LpLi microglia in apoptosis in cortex in mouse models were significantly higher than that frequency of Tp and TpTi microglia in apoptosis in mouse models detected, strongly suggesting that microglia from Lp and LpLi mice were more sensitive to apoptosis in vivo (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⁺ microglia and the neurosupportive, apoptotic sensitive, and IL-10- and TGF-b-biased CXCR3⁺Irg1^- microglia. However, we still need more direct evidences to show the functions of neurosupportive CXCR3⁺Irg1^- and neurotoxic CCR9⁺Irg1⁺ microglia in vivo. Moreover, we still need to collect more evidences on how CCR9⁺Irg1⁺ and CXCR3⁺Irg1^- microglia function as a neuron killer and supporter in vivo.

Many intrinsic factors in the neurons of central nervous system control and elaborate the immune functions of activated microglia. The membrane-bound and secreted factors from neurons and microglia, such as cytokines and chemokines, can control the immune activity in the central nervous system [7, 9, 27 - 30]. For example, the cytokines and chemokines play an important role of the communication and effector functions of microglia [28, 31]. The expression levels 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 up-regulated during T-cell development [32, 33]. A considerable number of investigations has shown that pair of CCR9/CCL25 is an important role for the T cell development, homing, homeostasis, particularly, mucosal T cells [34, 35]. In our previous study, CCR9-expressing microglia is majority of cell population of the Tp and TpTi microglia in mouse models, and CXCR3-expressing microglia is majority population of Lp and LpLi microglia in mouse models [5]. Two distinctive subsets of the microglia from the two different mouse models contribute distinctive roles in pathogenesis of neuronal degeneration or regeneration in central nervous system. Particularly, two distinctive subsets of the CXCR3⁺Irg1^- and CCR9⁺Irg1⁺ microglia have distinctive secreting profiles in cytokines and chemokines as well as expressions of chemokine receptor in respect to neuronal killer and neuronal supporter. In the present study, we have provided with more direct evidence to document the effects of neurosupportive CXCR3⁺Irg1^- and neurotoxic CCR9⁺Irg1⁺ microglia in Lp and LpLi mouse models as well as Tp and TpTi mouse models in vivo. Moreover, we have also observed that ligation of CCR9 by CCL25 can directly cause phosphorylation of Irg1 ex vivo in the freshly isolate Tp and TpTi CCR9⁺Irg1⁺ microglia from mouse models (Fig. 7). However, how CCR9/CCL25/Irg1 pathway contributes to microglia-involved regeneration and death of neurons in vivo should be further investigated.

Acknowledgement(s)

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.).

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