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The Journal of Immunology, 2006, 177: 3644-3656.
Copyright © 2006 by The American Association of Immunologists, Inc.

Different Neurotropic Pathogens Elicit Neurotoxic CCR9- or Neurosupportive CXCR3-Expressing Microglia1

He Li2,*,{dagger}, Zhou Gang2,*,{ddagger}, He Yuling2,*,{ddagger}, Xie Luokun2,*, Xiong Jie*,¶, Lei Hao||,#, Wei Li||, Hu Chunsong**, Liu Junyan§, Jiang Mingshen{dagger}, Jin Youxin, Gong Feili{dagger}{dagger}, Jin Boquan* and Tan Jinquan3,*,{ddagger},§

* Department of Immunology and {dagger} Department of Parasitology, Institute of Allergy and Immune-Related Diseases, {ddagger} Laboratories of Neuroscience, and § Allergy and Clinical Immunology, Center for Medical Research, Wuhan University School of Medicine, Wuhan, People’s Republic of China; The State Key Laboratory of Molecular Biology and Neuroscience, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, People’s Republic of China; || The State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, People’s Republic of China; # Key Laboratory of Colloid, Interface Science and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, People’s Republic of China; ** Department of Immunology, College of Basic Medical Sciences, Anhui Medical University, Hefei, People’s Republic of China; {dagger}{dagger} Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; and * Department of Immunology, Fourth Military Medical University, Xian, People’s Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-{alpha}; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+Irg1– microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-beta. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1– microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1– cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer’s disease and AIDS dementia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microglia make up the innate immune system of CNS and are key cellular mediators of neurodegenerative and neuroinflammatory processes. Microglia are now recognized as the prime components of an intrinsic brain immune system (1). Although their immune functions are "down-regulated" in healthy CNS, microglia are rapidly activated upon inflammatory processes, chronic brain diseases, or brain injury (2, 3). Activated microglia, the first cells in the CNS to respond to neuronal damage, are able to exert two opposing functions—promoting neuronal regeneration and killing neurons—by means of a variety of mechanisms (2, 4). Exerting one or the other function is largely determined by the particular circumstances that evoke microglial activation (2). However, the specific nature of any such constructive or destructive mechanisms remains nebulous.

Unlike others, the CNS is an immunologically privileged organ because of a relatively impermeable blood-brain barrier and an immunosuppressive microenvironment to limit immune cells entry and function. However, microglia may direct initial responses to neurotropic pathogenesis (5). The proximity of microglial and neuronal membranes is necessary to facilitate cell-cell communication by means of signaling factors—diffusible molecules and surface receptors such as chemokines and their receptors (6). A number of CC and CXC chemokines/receptors are believed as mediators of CNS development and pivotal players in the pathogenesis of immune-mediated neurodegenerative neuroinflammatory and diseases in the CNS (7). Importantly, CXCR3/CXCL10 signaling is crucial in microglia recruitment and an essential element for neuronal reorganization (6, 8). Although CCR9/CCL25 signaling is important for the homing, development, homeostasis, and resistance to apoptosis of T cells (9), there is so far no report on functional expression of CCR9 on microglia.

The immunoresponsive gene 1 (Irg1),4 an LPS-inducible gene (10), remains unclear in its expression pattern and biological functions (11).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animal models

Five- to 6-wk-old female C57BL/6 were obtained from The Jackson Laboratory and maintained in a pathogen-free environment in the Animal Research Institute, Medical Research Center, Wuhan University School of Medicine. Unless otherwise stated, the following animal models were used throughout in the study: mice were primed with three intracranially injections of 8 µg of Toxoplasma gondii (DX strain) soluble tachyzoite Ag (STAg) (termed Tp) (12) or primed with three intracranially injections of 0.1 µg of recombinant nucleoprotein immunodominant domain of lymphocytic choriomeningitis virus (rLCMVNP) (termed Lp) (13) in biweekly intervals. Two weeks after the last priming, Tp mice were intracranially infected (rechallenged) with a single dose of five cysts of T. gondii (sublethal, low-virulent DX strain, termed TpTi); Lp mice were intracranially infected with a single dose of 100 PFU of LCMV (sublethal, clone 13, termed LpLi) (14, 15); some Tp mice were cross-infected with LCMV (termed TpLi); some Lp mice were cross-infected with T. gondii (termed LpTi); and unprimed mice were intracranially infected with a single dose of T. gondii (termed Ti) or LCMV (termed Li). The mortality of mice was monitored semidiurnally for 55 days and was used to evaluate the clinical severity of infections of neurotropic pathogens. All animals were deeply anesthetized with the ketamine mixture before sacrifice. The experimental procedures were approved by the Animal Care and Use Committee of Wuhan University.

Flow cytometry

For microglia analysis, the cortex or hippocampus was microdissected from mice at indicated specific time intervals. Single-cell suspensions of cortex or hippocampus were prepared using a 70-µm mesh, followed by collagenase/DNase (Sigma-Aldrich) digestion and Percoll (Pharmacia) gradient separation. To avoid contamination of lymphocytes during flow cytometry, CD3+ and CD19+ lymphocytes were positively depleted from cell suspensions using anti-CD3 and anti-CD19 mAb-labeled magnetic Dynabead assay (Dynal Biotech). Both anti-mouse CD11b (Mac-1; Leinco Technologies) and CD45 (DakoCytomation) were used to stain the microglia (6, 8). Cells were stained with appropriate secondary fluorescence-labeled Abs. For detection of CCR9, CXCR3, or Irg1, a third color was stained by goat anti-mouse CCR9 pAb (E-15; Santa Cruz Biotechnology) or goat anti-mouse CXCR3 pAb (SC-6226; R&D Systems) or rabbit anti-mouse Irg1 (generated in-house), respectively. In some cases, four-color staining of CD11b-CD45-CD3-CCR9 or -CXCR3 in cell suspension was used to distinguish microglia populations from T cells and detection CCR9 and CXCR3 expression on different cell populations. An intracellular immunofluorescence staining procedure of IntraPrep (Coulter-Immunotech) was used to permeabilize cells in Irg1 staining. For detection of microglial and neuronal apoptosis, the microglia were purified using the high-gradient magnetic CD11b/Mac1-labeling cell separation system (MACS; Miltenyi Biotec) (8). Apoptosis of microglia was evaluated by binding assay of propidium iodide (1 µg/ml) and FITC-labeled annexin V (BD Clontech) (16). Neuron cultures were derived from fetal mouse cerebral cortices (17). Neuron-microglia cocultures (culture inserts) were maintained for 48 h (17). After treatment, culture inserts containing microglia were removed, and neurons were stained by the TUNEL method (Phoenix Flow Systems) (18). Data were acquired using a flow cytometer (COULTER XL; Coulter).

Histological analysis

Cerebral cortex and hippocampal tissues of different mouse models were fixed in 10% formalin and paraffin processed. Five-micrometer histological sections were stained with H&E and photographed. For immunohistochemistry staining (6, 8), free-floating vibratome sections were permeabilized. Endogenous peroxidases were blocked by 0.3% H2O2. Sections were incubated in blocking solution containing 5% BSA, followed by overnight incubation at 4°C with primary Ab (anti-CD11b/Mac-1, anti-CCR9, anti-CXCR3, or anti-Irg1). Appropriate biotinylated secondary Ab was applied for 2 h at room temperature. Abs were then visualized with avidin-biotin-peroxidase complex (DakoCytomation). For quantification of positive cells, at least nine sections per animal and four randomly selected regions in each section were analyzed. All slides were viewed on an Olympus Van Ox microscope and evaluated in a blinded fashion by two independent experts. For immunofluorescence digital confocal microscopy (9), the purified cells were spun down on a slide, fixed, immersed with 1% BSA blocking buffer, added with primary Ab, and incubated overnight at 4°C, followed by fluorescence-labeled secondary Ab fluorescence labeling. Confocal microscopy analysis was performed using a confocal laser scanning system and an inverted microscope (LSMSIO; Zeiss).

Western and Northern blotting

For coimmunoprecipitations and phosphorylated-protein detection, cells were washed extensively and lysed in lysis buffer (19). Cell lysates were centrifuged (10,000 rpm for 10 min, 4°C), and the supernatants were recovered. Cell lysates were precleared three times with 20 µl of protein A-Sepharose beads and were mixed with specified Ab (anti-Irg1) for 3 h at 4°C under constant agitation. Immune complexes were allowed to bind to 20 µl of protein A-Sepharose beads overnight, beads were washed three times with lysis buffer, and immunoprecipitates were separated in 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Filters were blocked with 5% nonfat milk in blocking buffer and incubated with specific rabbit anti-phosphorylated proteins Ab (Zymed Laboratories) for 2 h, followed by HRP-labeled secondary Ab (Amersham Biosciences) visualizing procedure and autoradiography. For Western blot analysis (20), protein concentration in cell lysates was measured by Bio-Rad protein assay. Protein (~40 µg) was loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after electrophoresis, and incubated with the appropriate Abs (CCR9, CXCR3, and Irg1) at 0.5 µg/ml. The membrane was blocked in 5% BSA-TBS, followed by secondary Ab visualizing procedure and autoradiography (19). For mRNA detection, 5 µg of pooled total RNA samples was electrophoresed under denaturing conditions, followed by blotting onto Nytran membranes, and cross-linked by UV irradiation as described previously (21). Specific appropriate cDNA probes labeled by [{alpha}-32P]dCTP were hybridized overnight with membranes followed by intensively washing with 0.2x SSC before being autoradiographed.

Real-time quantitative RT-PCR

Total RNA was purified from microglia or brain tissues. All real-time quantitative RT-PCR were performed as described elsewhere (22). Briefly, total RNA was purified from microglia or brain tissues. Total RNA was prepared by using the Quick Prep total RNA extraction kit (Pharmacia Biotech). The RNA was reverse transcribed by using oligo(dT)12–18 and Superscript II reverse transcriptase (Invitrogen Life Technologies). DNA in the tissues from T. gondii-infected animals tissues were extracted using the Qiamp tissue kit (Qiagen). The real-time quantitative PCR was performed with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems) by using the SYBR Green PCR Core Reagents kit (Applied Biosystems) according to the manufacturer’s instructions. Primers used for real-time quantitative PCR were shown in Table I.


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  		Table I. The sequences of senses and antisenses for mRNA detection in real-time quantitative RT-PCR assay

 
ELISA analysis

Briefly, cytokine and chemokine proteins were measured in conditioned medium from cultures of microglia using ELISA kits (Promo Cell) (23). Culture supernatant was collected after incubation for the time indicated from microglia culture. Media samples were first cleared at 2000 rpm for 5 min and then assayed for cytokine and chemokines according to the manufacturer’s instructions. The experiments were performed six times for each group, and each sample was assayed in triplicate.

Cell transient transfection

Purified microglia were transiently transfected with plasmids encoding CCR9, CXCR3, and Irg1 using the Amaxa nucleofection technology (Amaxa) with optimization according to the manufacturer’s instructions. Briefly, cells were resuspended in solution from nucleofector kit V, following the Amaxa guidelines for cell transfection. One hundred microliters of 3 x 106 cell suspension mixed with 0.25 or 2.5 µg of cDNA was transferred to the provided cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa).

Small interfering RNA (siRNA) gene knockdown assay

The design of siRNA was based on the characterization of siRNA (24). The siRNA were synthesized in 2'-deprotected, duplexed, desalted, and purified form by Dharmacon Research. The sense of mouse HO-1 siRNACCR9 was as follows: 5'-GTCATCCAAGCACAAGGCCCT-3'; and siRNAIrg1, 5'-GTATCATTCGGAGGAGCAAGA-3'. For in vivo studies, each mouse was anesthetized and then given intranasal HO-1 siRNA (8 mg/kg/day) or equivalent doses of mismatched control siRNA duplex in a volume of 50 µl. The administration began 2 wk before other experimental procedures and continued till the end of experiments.

Chemotaxis and nitrite quantification assays

Migration of microglia in response to CCL25 and CXCL10 (R&D Systems) was determined as described earlier (25). NO2– in culture supernatants was measured to assess total NO production in microglial cells as described earlier (16). Briefly, 50 µl of sample aliquots of culture supernatants from microglial cells was mixed with 50 µl of Griess reagent (1% sulfanilamide-0.1% naphthylethylenediamine dihydrochloride-2% phosphoric acid) with adding nitrate reductase (0.003 U of Aspergillus species; Sigma-Aldrich) in plates and incubated at 25°C for 10 min for measurement of for total NO levels. The OD550 was measured on a microplate reader.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
T. gondii- and LCMV-priming differentially elicit microglia

To explore which factor(s) determined differential behavior of activated microglia for neuronal regeneration or degeneration, we established different animal models to induce activated microglia in vivo (see Materials and Methods). Unexpected mortalities in TpTi and TpLi mice were observed after a sublethal dose infection. There was no death case in Tp, Ti, Lp, Li, and LpLi mice and a minor mortality in LpTi mice (Fig. 1A). Significant increased microglial frequencies in cortex in TpTi and LpLi mice were observed (Fig. 1B). There was a significant and rapid increase of microglia at the early stage in TpTi mice and a late increase in LpLi mice during pathogenesis (Fig. 1C). By H&E staining and immunohistochemistry, a higher frequency of microglia, severe edema, a large number of necrotic neurons, and many "cap-like" microglia-neuron bodies were seen observed in cortex of TpTi mice. Meanwhile, a higher frequency of microglia, a large number of infiltrating lymphocytes, and microglia-lymphocyte "rosette-like" bodies were found in the cortex of LpLi mice (arrows, Fig. 1D). T. gondii DNA was not significantly altered in the brain of TpTi mice in comparison with Ti mice but significantly decreased in LpTi mice. Similar data were obtained in the microglia-infiltrating inflammation-reactive hippocampus region from different mouse models (data not shown). LCMV mRNA significantly decreased in the brain of LpLi mice in comparison with that of Li mice, whereas LCMV mRNA slightly increased in TpLi mice (Fig. 1E). T. gondii DNA and LCMV mRNA burden were examined in other vital organs (liver, spleen, kidney, lung, heart, and gut). The pathogens were at low or undetectable levels in these organs, indicating that the animal death was not contributed by spreading to other organs of pathogen infection (data not shown). Thus, T. gondii Ag intracranial priming exacerbated brain damage and caused death (TpTi and TpLi mouse models). LCMV intracranial priming showed significant protective effects (LpLi and LpTi mouse models). T. gondii priming-induced microglia were rapidly reactive, directly attacked neurons, and were unable to clean pathogens when the CNS was infected; LCMV priming-induced microglia were relatively slowly reactive, attracted lymphocytes, and were able to clean infected pathogens. These clues led us to search for possibility of distinctive subsets of microglia.


Figure 1
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  		FIGURE 1. Analyses of microglial behaviors from different animal models. A, Mortality of mice with different treatments (n = 10). {square}, Tp; {blacksquare}, TpTi; {circ}, Ti; or •, TpLi (left panel); {square}, Lp; {blacksquare}, LpLi; {circ}, Li; or •, LpTi (right panel). B, Flow cytometric analysis of microglia from normal (NML), Tp, TpTi, Ti, TpLi, Lp, LpLi, Li, and LpTi mice. The animals were sacrificed either at end of the priming procedure or at day 10 postinfection. Lymphocyte-depleted single-cell suspensions from cortexes were prepared and stained. Microglia were identified on the basis of expression of CD11b/Mac1 and CD45 double positive. Data were representatives of eight animals per group. C, Analyses of the number of microglia in slices from cortex of NML ({diamondsuit}), TpTi ({blacksquare}), TpLi (•), LpLi ({square}), or LpTi ({circ}) mice and sacrificed at days as indicated. CD11b/Mac1+ microglial numbers were represented as mean ± SD per high-power field (hpf) and n = 5. *, p < 0.001, TpTi vs TpLi, LpTi, or LpLi at day 6; *, p < 0.01, LpLi vs TpLi or LpTi at day 30. D, Morphology and distribution of CD11b/Mac1+ microglia (brown) infiltrating inflammation-reactive cortex determined by H&E staining and immunohistochemistry (n = 6). Arrows, Necrotic neurons and "cap-like" microglia-neuron bodies (TpTi) and lymphocyte-infiltrating and microglia-lymphocyte "rosette-like" bodies (LpLi). The insets were isotype controls. E, Levels of T. gondii DNA and LCMV RNA in the inflammation-reactive cortex of Ti, TpTi, or LpTi mice (left panel), Li, LpLi, or TpLi mice (right panels) determined by real-time quantitative PCR. Mice were sacrificed at day 10 postinfection with different neurotropic pathogens. Data were mean ± SD (n = 5). *, p < 0.001, LpTi vs Ti or TpTi; LpLi vs Li or TpLi. LCMV RNA levels were verified by Northern blot assay (lower right panel).

 
Microglia from different animal models differentially express CCR9 and CXCR3

To further characterize the microglia elicited from different mouse models, we analyzed the all known chemokine receptor expressions in microglia from different mouse models (Table II). The microglia in cortex in Tp and TpTi mice expressed a high level of CCR9, whereas the microglia elicited from Lp and LpLi mice expressed a high level of CXCR3 (Fig. 2A). T. gondii infection mainly induced CCR9 expression (Ti mice), whereas LCMV infection mainly induced CXCR3 expression (Li mice). Similar data were obtained in the microglia-infiltrating inflammation-reactive hippocampus region from different mouse models (data not shown). As it is presented in Fig. 2, B–D, by real-time RT-PCR, Western and Northern blots confirmed that at both mRNA and protein levels, CCR9 expression was dominant in microglia of Tp and TpTi mice, whereas CXCR3 was expressed at a high level in Lp and LpLi mice. CCL25 induced strong chemotactic migration of microglia from Tp and TpTi mice but not from Lp and LpLi mice. CXCL10 induced strong chemotaxis of microglia from Lp and LpLi mice but not from Tp and TpTi mice (Fig. 2E). We applied four-color flow cytometry in cell suspension with combined staining of CD11b-CD45-CD3-CCR9 or -CXCR3 to distinguish microglia populations from T cells. Within CD11b+CD45+ microglia populations, CCR9 was highly up-regulated in Tp and TpTi mice, and CXCR3 was highly up-regulated in Lp and LpLi mice, in comparison with normal mice. Meanwhile, within CD3+CD45+ T cell populations, both CCR9 and CXCR3 were expressed at very low levels (data not shown). The results confirmed that up-regulation of CCR9 and CXCR3 expression was indeed on microglia.


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  		Table II. The real-time detection of mRNA of chemokine receptors on microglia from different mouse modelsa

 

Figure 2
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  		FIGURE 2. CCR9 and CXCR3 expression on microglia from different mouse models. A, Triple-color flow cytometric analyses of CCR9 and CXCR3 on microglia from NML, Tp, Ti, TpTi, TpLi, Lp, Li, LpLi, and LpTi mice. The animals were sacrificed as described in Fig. 1B. Red curves, CCR9 staining; blue curves, CXCR3 staining; gray curves, isotype Ab controls. The numbers were indicating the percentages of the positive cells. Data were representatives of eight animals per group. B, Morphology and distribution of CCR9- or CXCR3-positive (brown) microglia-infiltrating inflammation-reactive cortex determined by immunohistochemistry (n = 6). Magnifications, x400. The insets were isotype controls. C, The microglia-infiltrating inflammation-reactive cortex were photographed by immunofluorescence digital confocal microscopy (n = 6). Magnification, x1200. Bar, 12 µm. The arrows: negative (N), CCR9+, CXCR3+, and CCR9+CXCR3+ (DP) cells (all left panels). The insets were isotype controls. The percentages (all right panels) of different microglia: negative, gray; CCR9+, red; CXCR3+, green; and CCR9+CXCR3+, yellow. The experiments in each group were independently repeated twice. D, Expression levels of CCR9 and CXCR3 in microglia infiltrating inflammation-reactive cortex determined by Northern and Western blot analyses (left panels) and real-time quantitative RT-PCR (right panels) in purified microglia from cortex (n = 6). *, p < 0.001, Tp or TpTi vs others (CCR9); Lp or LpLi vs others (CXCR3). E, Chemotaxis of microglia infiltrating inflammation-reactive cortex toward CCL25 (left panel) and CXCL10 (right panel). Tp ({blacksquare}) or Lp mice ({square}) were scarified at time described in Fig. 1B. All results were expressed as chemotactic index (C.I. ± SD; n = 6). *, p < 0.001, Tp vs Lp.

 
Microglia from different mouse models function differentially

In culture, the number of apoptotic microglia was very low in Tp and TpTi mice but very high in Lp and LpLi mice in comparison with those in NML mice (Fig. 3A). The numbers of apoptotic microglia in cortex in Lp and LpLi mice were significantly higher than that in Tp and TpTi mice detected by TUNEL assay in vivo, indicating that Lp and LpLi microglia were sensitive to apoptosis (data not shown). In coculture of neuron-microglia, the numbers of apoptotic neurons were very high in Tp and TpTi mice and very low in Lp and LpLi mice in comparison with NML mice (Fig. 3B). Lp microglia inhibited apoptotic effect of Tp microglia on neurons in vitro (data not shown). Microglia from different mouse models (Tp, TpTi, Lp, and LpLi mice) produced similar levels of IL-2, IFN-{gamma}, and IL-4 in culture at mRNA level (Fig. 3C). Microglia from Tp and TpTi mice expressed significant higher level of TNF-{alpha}, whereas microglia from Lp and LpLi mice produced significant higher levels of IL-10 and TGF-beta at mRNA level (Fig. 3C). Microglia from Tp and TpTi mice expressed significant higher level of CCL25. Microglia from Lp and LpLi mice produced significant higher level of CX3CL1 and CXCL10 at mRNA level (Fig. 3C). There was no difference in production of CCL5 and CXCL12. Using ELISA, we confirmed the similar pattern of cytokine and chemokine expressions in microglia from different mouse models at protein level (Fig. 3D). In culture without LPS, microglia from Tp and TpTi mice produced 3- to 5-fold higher nitrite than those in NML, Lp, and LpLi mice. In culture with LPS, microglia from Tp, TpTi, Lp, and LpLi mice secreted identical amounts of nitrite (Fig. 3E). NO inhibitor N{omega}-nitro-L-arginine methyl ester significantly inhibited apoptotic effect of Tp and TpTi microglia on neurons in vitro (data not shown). Moreover, mRNA of brain-derived neurotrophic factors, a member of the neurotrophin family, was at a low level in NML microglia and did not alter in Tp, TpTi, Lp, and LpLi mice (data not shown). Thus, Tp and TpTi mouse model elicited the microglia that were expressing CCR9 at a high frequency, resistant to apoptosis, neurotoxic, expressing a high level of TNF-{alpha} and autoligand CCL25, and high NO productive; the microglia from Lp and LpLi mice were frequently highly expressed CXCR3, sensitive to apoptosis, neurosupportive, producing a high level of IL-10, TGF-beta, autoligand CXCL10, and a low level of NO.


Figure 3
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  		FIGURE 3. Functions of microglia from different mouse models. A. Flow cytometric analysis of apoptotic microglia following stimulation with (red curves) or without LPS (green curves) (n = 8). The microglia were first purified from mice as described Fig. 1B, and cultured for 2 days and stained. T. gondii inhibitor pyrimethamine (1.0 µg/ml, Sigma) and LCMV inhibitor ganciclovir (GCV, 1.0 µg/ml, Roche Diagnostics) were added into the cultures to avoid direct effects of pathogens. The microglia were analyzed by flow cytometry for PI and FITC-conjugated annexin V (shown, left panel). Data for apoptotic microglia were mean ± SD with LPS stimulation (right panel). *, p < 0.001, NML, Lp, or LpLi vs Tp or TpTi. B. Flow cytometric analysis of apoptotic neurons. The neurons were derived from cerebral cortices of fetal (embryonic day 17) mice (n = 8), and cocultured with purified microglia infiltrating inflammation-reactive cortex from mice in the presence (red curves) or absence of LPS (green curves) for 48h with pyrimethamine and ganciclovir in culture. The neurons were analyzed for FITC-conjugated BrdU. The data for apoptotic neurons were from a single experiment (left panel) or mean ± SD (right panel) with LPS stimulation. *, p < 0.001, Tp or TpTi vs NML, Lp, or LpLi. C, Levels of cytokine (upper panels) and chemokine (lower panels) mRNAs of microglia-infiltrating inflammation-reactive cortex determined by real-time quantitative RT-PCR. The microglia were purified as described in Fig. 1B (n = 8) and cultured at presence of LPS for 6 h with pyrimethamine and ganciclovir in system. Upper leftmost panel, {square}, IL-2; {blacksquare}, IFN-{gamma}. *, p < 0.001, Tp or TpTi vs NML, Lp, or LpLi for TNF-{alpha}; *, p < 0.01–0.001, NML, Lp, or LpLi vs Tp or TpTi for IL-10 and TGF-beta; *, p < 0.001, Lp or LpLi vs NML, Tp or TpTi for CX3CL1 and CXCL10; *, p < 0.001, Tp or TpTi vs NML, Lp, or LpLi for CCL25. D, Cytokine and chemokine production of microglia from different mouse models (NML, Tp, TpTi, Lp, and LpLi mice). Levels of cytokine (upper panels) and chemokine (lower panels) in culture supernatant of microglia infiltrating inflammation-reactive cortex determined by ELISA as described in Materials and Methods. The animals were sacrificed either at end of priming procedure or at day 10 postinfection. The microglia were purified using the high-gradient magnetic CD11b/Mac1-labeling cell separation system (n = 4) and cultured at the presence of LPS for 6 h with pyrimethamine and ganciclovir in system. Upper leftmost panel, {square}, IL-2; {blacksquare}, IFN-{gamma}. *, p < 0.001, Tp or TpTi vs NML, Lp, or LpLi for TNF-{alpha}; *, p < 0.01–0.001, NML, Lp, or LpLi vs Tp or TpTi for IL-10 and TGF-beta; *, p < 0.001, Lp or LpLi vs NML, Tp or TpTi for CX3CL1 and CXCL10; *, p < 0.001, Tp or TpTi vs NML, Lp, or LpLi for CCL25. E, Total NO production of microglia following stimulation with ({blacksquare}) or without LPS ({square}) (n = 6). *, p < 0.001, Tp or TpTi vs NML, Lp, or LpLi in absence of LPS; *, p < 0.001, presence vs absence of LPS in Lp or LpLi microglia.

 
Microglia from Tp and TpTi mice selectively express Irg1

A report on that >63-fold up-regulation of Irg1 in activated microglia in vitro (26) led us to examine the Irg1 expression in cortex from different mouse models. Microglia from Tp and TpTi mice intracellularly expressed very high levels of Irg1 but not in the cells from NML, Lp and LpLi mice (Fig. 4A). The observations were confirmed by immunohistochemistry (Fig. 4B), by immunofluorescence digital confocal microscopy (Fig. 4C, Tp mice data not shown), and by Northern and Western blots, and real-time quantitative RT-PCR (Fig. 4D). Irg1-expressing microglia were also found in hippocampus region of Tp and TpTi mice (data not shown).


Figure 4
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  		FIGURE 4. Expression of Irg1 in microglia from different mouse models. A, Triple-color flow cytometric analysis of Irg1 in microglia from NML, Tp, TpTi, Lp, and LpLi mice. Lymphocyte-depleted single-cell suspensions from cortices were prepared and stained as described in Materials and Methods. Gray curves, isotype Ab controls. The numbers showed Irg1+ cells. Data were representatives of eight animals per group. B, Distribution of Irg1-positive microglia-infiltrating inflammation-reactive cortex determined by immunohistochemistry. The arrows were indicating Irg1-expressing microglia (brown). The experiments in each group (n = 6) were independently repeated twice. Magnifications, x1000. The insets were isotype controls. C, Distribution of Irg1+ microglia-infiltrating inflammation-reactive cortex determined by immunofluorescence digital confocal microscopy. The microglia in cortex from TpTi mice were purified and stained. Magnification, x1200. Bar, 12 µm. (i) was isotype control. D, Expression levels of Irg1 in microglia-infiltrating inflammation-reactive cortex from mice determined by Northern and Western blot analyses (upper and middle panels) and real-time quantitative RT-PCR (lower panel). The illustrated data are from a single representative experiment of each group animals (n = 6). The mRNA of Irg1 in purified microglia from cortex was determined by real-time quantitative detection of RT-PCR. The showing bars were mean ± SD of six experiments in each group. *, p < 0.001, Tp or TpTi vs NML, Lp, or LpLi.

 
Neurotoxic microglia function via CCL25/CCR9/Irg1 pathway activation

We cotransfected CCR9, CXCR3, and Irg1 into purified microglia from fetal mice cerebral cortices. Levels of phosphorylated-Irg1 proteins were dependent on transfected amount of pan Irg1 proteins and ligation of CCR9/CCL25. CXCR3/CXCL10 ligation induced no phosphorylated-Irg1 proteins (Fig. 5A1). The levels of phosphorylated-Irg1 were dependent on the transfected amount of CCR9 proteins (Fig. 5A2) but not on the transfected amount of CXCR3 protein (Fig. 5A3). CCL25 could also directly induce Irg1 phosphorylation in freshly isolated CCR9+Irg1+ microglia from Tp and TpTi mice (data not shown). We cotransfected CCR9 and CXCR3 into purified microglia in the absence or presence of Irg1 transfection. Real-time RT-PCR data revealed that CCL25 ligation caused increased levels of TNF-{alpha} mRNA (Fig. 5B1) and reduced levels of IL-10 and TGF-beta mRNA in an Irg1-transfection-dependent manner (Fig. 5B2). CCR9/CCL25 ligation could induce higher NO production in transfected microglia than CXCL10/CXCR3 in an Irg1-transfection-dependent manner (Fig. 5C). CCR9/CCL25 ligation enhanced the resistance to TNF-{alpha}-induced apoptosis of microglia in an Irg1 transfection-dependent manner (Fig. 5D). CCR9/CCL25 ligation enhanced the neurotoxicity of microglia to cause neuron apoptosis in an Irg1 transfection-dependent manner (Fig. 5E). Thus, by means of CCR9/CCL25/Irg1 pathway, activated microglia produce higher levels of TNF-{alpha} and lower IL-10 and TGF-beta, along with enhanced resistance to apoptosis and increased neurotoxicity.


Figure 5
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  		FIGURE 5. CCL25/CCR9 selectively and functionally activates Irg1 protein in microglia. A, Normal microglia purified from fetal mice cerebral cortices were cotransfected with vectors encoding CCR9, CXCR3, and Irg1 (600 ng each constantly; or 200, 400, and 600 ng, respectively) as indicated (A1, A2, and A3). Cells were pretreated at absence or presence of CCL25 and/or CXCL10 (each 100 ng/ml) before cell lysis. Irg1 proteins were immunoprecipitated from the cell lysates for detection of levels of phosphorylation using a rabbit anti-phosphorylated proteins Ab. B. The mRNA levels of TNF-{alpha} (B1), IL-10 ({square}), and TGF-beta ({blacksquare}) (B2) in microglia-transfected CCR9 and CXCR3 at absence (upper panels) or presence (lower panels) of Irg1 transfection determined by real-time quantitative RT-PCR. Cells were pretreated at absence or presence of CCL25 and/or CXCL10 (each 100 ng/ml) for 24 h before cell lysis. LPS was used as positive controls. The showing bars were mean ± SD (n = 8). *, p < 0.001. C, NO production of microglia-transfected CCR9 and CXCR3 at absence (upper panel) or presence (lower panel) of Irg1 transfection (n = 8). Cells were stimulated without or with CCL25 and/or CXCL10 (each 100 ng/ml). LPS was used as positive controls. D and E, Flow cytometric analysis of apoptotic microglia (D) and neurons (E). The procedures were described in Fig. 3, A and B. The microglia transfected CCR9 and CXCR3 at absence (upper panels) or presence (lower panels) of Irg1 transfection. Exogenous TNF-{alpha} (20 ng/ml) was used as an apoptotic inducer. Data for apoptotic microglia (D) and apoptotic neurons (E) were mean ± SD (n = 8, *, p < 0.001).

 
CNS-resident microglia and blood-derived macrophages function differentially in vivo

To determine which cells, CNS-resident microglia or CNS-infiltrating macrophages (8, 27), might be a major responsive population in different mouse models, purified resting microglia and blood-derived macrophages (28) were transfected with CCR9 plus Irg1 or CXCR3 and subsequently primed with TSAg and rLCMVNP, respectively. CCR9+Irg1+-transfected microglia (intracranially) or CXCR3-transfected macrophages (i.v.) were infused into adult mice, respectively. Mice were then intracranially infected with T. gondii (Ti mice) or LCMV (Li mice). The results of cell frequency (Fig. 6, A and B) and proliferation (Fig. 6, C and D) showed that TSAg-primed CNS-resident CCR9+Irg1+ microglia dominantly functioned and highly proliferated in Ti mice, whereas both CXCR3+ CNS-resident microglia and blood-derived macrophages rapidly infiltrated into CNS and proliferated in a specific Ag priming-dependent manner in Li mice. Thus, CCR9+Irg1+ neurotoxic microglia, often seen in Tp and TpTi mice, were mainly generated from CNS-resident resting microglia; CXCR3+Irg1– neurosupportive microglia, often seen in Lp and LpLi mice, would be generated from both these infiltrating blood-derived vascular macrophages (might be also perivascular macrophages) (29) and CNS-resident resting microglia.


Figure 6
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  		FIGURE 6. Analysis of CNS-resident or blood-derived CCR9+Irg1+ or CXCR3+ microglia/macrophages in Ti or in Li mice. Normal purified microglia from fetal mice cerebral cortices and purified F4/80+ blood-derived macrophages (27 ) (CI:A3-1; Abcam) were cotransfected with vectors encoding CCR9 and Irg1 (A and C) or CXCR3 (B and D) (600 ng each), then cultured at absence or presence of TSAg (1 µg/ml) or rLCMVNP (0.5 µmol; Ref. 17 ) for 48 h indicated as TSAg–, TSAg+, LCMV–, or LCMV+. Cells were labeled with CFSE (Molecular Probes). The 106 of transfected microglia (intracranially) or macrophages (i.v.) infused were into adult mice, respectively, indicated as (Microglia i.c.) and (Macrophage i.v.). After 24 h of infusion, the mice were intracranially infected with T. gondii DX strain (five cysts, Ti mice) (A and C) or LCMV clone 13 (100 PFU, Li mice) (B and D). Mice sacrificed at day as indicated. A and B, Triple-color flow cytometric analyses of CCR9 or CXCR3 on microglia/macrophage from Ti mice (A) or Li mice (C). Black curves, CCR9 or CXCR3 staining; gray curves, isotype Ab controls. The shown numbers were percentages of CCR9 or CXCR3-positive cells. Data were representatives of eight animals per group. The experiments in each group were independently repeated twice. B and D, Flow cytometric analyses of the intensity of CFSE on microglia/macrophage from Ti mice (C) or Li mice (D). Data were representatives of eight animals per group.

 
CCR9 and Irg1 siRNA alter neurotoxic and neurosupportive profiles of microglia in vivo

TpTi mice administered with sufficient doses (2 µg) of hairpin siRNACCR9, siRNAIrg1, or siRNAMIX were significantly knocked down expression of CCR9 and Irg1 during the process of establishment of TpTi mouse model (Fig. 7A) but not administrating low doses (0.02 µg) of the substances (data not shown). The high (Fig. 7A) or low (data not shown) dose of mismatched siRNA sequence (siRNAMIS) did not show any knocking-down effect. There were "cross-knocking-down" phenomena, e.g., siRNACCR9 also inhibited Irg1 expression, and vice versa (Fig. 7A). The possible explanation could be that CCR9 and Irg1 in the pathway had a close cross-taking, the expression and function of one would interfere with those of the other. The exact mechanism of the reciprocal inhibition should be subjected to further investigation. Data of flow cytometry (Fig. 7B) and immunohistochemistry (Fig. 7C) revealed that siRNAMIX administration significantly knocked down CCR9 expression and up-regulated CXCR3 expression on microglia in cortex from TpTi mice. The similar results were also seen in TpTi mice administered with siRNACCR9 or siRNAIrg1 (data not shown). Mortality of TpTi mice with different administration revealed (Fig. 7D) that NS (data not shown) and siRNAMIS did not change the pattern of TpTi mice death. The administrations of siRNACCR9, siRNAIrg1, or siRNAMIX significantly decreased mortality in TpTi mice. Moreover, administrations of siRNACCR9, siRNAIrg1, or siRNAMIX suppressed the levels of T. gondii DNA in cortex of TpTi mice (Fig. 7E).


Figure 7
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  		FIGURE 7. Effects of siRNACCR9 and/or siRNAIrg1 on TpTi mice. A, CCR9 and Irg1 expression in microglia from TpTi mice determined by Northern and Western blot analyses (left panels) and real-time quantitative RT-PCR (right panels). Mice were treated with normal saline (NS), siRNACCR9, siRNAIrg1, siRNACCR9 plus siRNAIrg1 (siRNAMIX), or mismatch siRNA (siRNAMIS) along with priming and infection with T. gondii and scarified at day 10 of infection (n = 10). The showing bars for CCR9 and Irg1 mRNA were mean ± SD. *, p < 0.01–0.001, siRNACCR9, siRNAIrg1, or siRNAMIX vs NS or siRNAMIS for CCR9 and Irg1. B, Triple-color flow cytometric analysis of CCR9 and CXCR3 on microglia from individual TpTi animals (n = 8). The animals were treated with siRNAMIX and sacrificed at day 14 postinfection. Red curves, CCR9 staining; blue curves, CXCR3 staining; gray curves, isotype Ab controls. C, Distribution of CCR9+ or CXCR3+ microglia-infiltrating inflammation-reactive cortex from TpTi mice (n = 8) determined by immunohistochemistry. The animals were treated with siRNAMIX and sacrificed at day 14 postinfection. Magnifications, x400. The insets were isotype controls. D, Mortality of mice with different treatments. The animals were treated with NS (data not shown), siRNACCR9 (•), siRNAIrg1 ({square}), siRNAMIX ({circ}), or siRNAMIS ({blacksquare}) along with priming and infection with T. gondii. Mice (n = 10) were monitored daily for development of clinical symptoms and survival. E, Levels of T. gondii DNA in cortex of TpTi mice treated with NS, siRNACCR9, siRNAIrg1, siRNAMIX, or siRNAMIS. T. gondii DNA determined by real-time quantitative PCR were mean ± SD (n = 10). *, p < 0.001, siRNACCR9, siRNAIrg1, or siRNAMIX vs NS or siRNAMIS.

 
Two types of microglia in CNS by priming with different neurotropic pathogens

We further examined effects on microglial elicitation in CNS using a series of vaccines (Ags) of neurotropic pathogens, including different intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury. The intracranial priming with T. gondii STAg (virulent RH strain), HIV vaccine (30), rTMEV-VP3, and Abeta25–35 significantly caused an elicitation of neurotoxic CCR9+Irg1+ microglia in CNS (Table III). Generally, in mice administered with the neurotropic pathogens causing chronic but irreversible neurodemyelinative or neurodegenerative diseases, such as AIDS, Alzheimer’s disease, Theiler’s murine encephalomyelitis virus-induced demyelinating disease, experimental autoimmune encephalomyelitis model, and some of the intracellular parasitic (T. gondii) infection, microglial elicitation was oriented to neurotoxic. In mice administered with the neurotropic pathogens causing acute but reversible neuroinflammatory diseases, such as most of viral and bacterial encephalitis and reversible brain lesions, the microglia were apparently developing into neurosupportive cell type.


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  		Table III. Effects on microglial derivation in CNS by priming with antigens or other procedures of different neurotropic pathogensa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Microglia are now recognized as the prime and major components of an intrinsic brain immune system in the CNS (1). Many researchers now consider this innate immune response in the brain to be a potentially pathogenic factor in a number of CNS diseases that lack the prominent leukocytic infiltrates of adaptive immune responses, but that do have activated microglia to initiate neuroinflammation and neurodegeneration (1, 4, 31). An important but yet unresolved question is whether microglial activation in vivo can be triggered by pathophysiological stimuli that are not neuron derived and whether such idiopathic activation of microglia can result in microglial neurotoxicity exerted toward healthy neurons (4). This issue is particularly relevant to understanding neurodegenerative diseases such as AIDS and Alzheimer’s disease, in which chronic CNS inflammation is believed to play a role in the pathogenesis (31). In the present study, we have used different Ags (pathogens)—STAg from T. gondii low-virulent DX strain, a representative of neurotropic intracellular parasite, and rLCMVNP of LCMV low-virulent clone 13, a representative of neurotropic chronic virus—to directly (intracranially injection) and differentially prime microglia. After priming, a single dose of T. gondii or LCMV have sublethally and intracranially been infected (rechallenged) in mice. These approaches allow us to investigate preactivated (primed) microglial behaviors in different neurotropic pathogenesis in vivo. T. gondii Ag and LCMV Ag priming have indeed elicited two different types of microglia (Figs. 1–4). We have further confirmed the observation by intracranially priming and rechallenging in mice with a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury (Table III). We therefore tentatively suggest to term the neurotoxic CCR9+Irg1+ cell pattern as killer microglia (kM) and the neurosupportive CXCR3+Irg1– cell pattern as supporter microglia (sM).

We have found that microglia in normal animals appear at very low frequency (Fig. 1, B–D). Most of the microglia in normal animals are CXCR3–CCR9–Irg1– (Figs. 2 and 4). They seem to be inactive with respect to their functions of production of cytokines and chemokines in vitro (Fig. 3). There is so far no significant marker found on their surface with regard to expression of chemokine receptors (Table. II). We have also obtained similar data in peripheral macrophages in normal animals (data not shown). However, are two subsets of microglia in CNS differentiated from a third phenotypic subset of inactive/undifferentiated microglia? Or, do they proliferate or migrate to the site of pathology and differentiate into one or the other subsets by the specific pathogen? We have found indirect evidence that CCR9+Irg1+ microglia are mainly derived from CNS-resident resting microglia; CXCR3+Irg1– microglia are derived from both these infiltrating blood-derived vascular macrophages and CNS-resident resting microglia (Fig. 6). The direct evidence is warranted for further confirmation of these hypotheses.

The microglial immune functions are controlled and elaborated by intrinsic factors in the CNS neurons. Both membrane-bound and secreted factors, such as cytokines and chemokines, possess the potential of controlling endogenous immune activity within the CNS (6, 8, 32, 33, 34, 35). Cytokines and chemokines constitute a substantial fraction of the microglial communication and effector system. TNF-{alpha}, IL-6, and IFN-{gamma} have been shown to be critical for protective actions as well as harmful outcomes of microglial engagement (33, 36). Chemokine receptor CCR9 expression is highly regulated during T cell development. Most immature DN thymocytes express no or low levels of CCR9 on their surface (37, 38). A considerable body of investigations has shown that CCR9/CCL25 is important for the homing, development, and homeostasis of T cells, particularly, mucosal T cells (39, 40). In the present study, we have documented that CCR9-expressing microglia is the dominant phenotype in Tp and TpTi mice, and CXCR3-expressing microglia is the predominant cell population in Lp and LpLi mice. To our knowledge, this is the first observation on expression of CCR9 on microglia in CNS. Furthermore, we have demonstrated that Tp and TpTi mouse model-induced CCR9+Irg1+ killer microglia express a high level of TNF-{alpha}, CCL25, and NO by means of CCR9/CCL25/Irg1 pathway activation; Lp and LpLi mouse model-induced CXCR3+Irg1– supporter microglia produce high levels of IL-10, TGF-beta, CXCL10, and relatively lower levels of NO (Figs. 3 and 5). Our data suggest that distinctive microglia can be induced by different neurotropic pathogens. Of different profiles in cytokine and chemokine release and chemokine receptor expression, killer and supporter microglia play distinctive roles in pathogenesis to degenerate or to regenerate CNS neuron. The direct evidence is still lacking to demonstrate neurotoxic and neurosupportive effects of CCR9+Irg1+ and CXCR3+Irg1– microglia in vivo. It is worthwhile to investigate whether there is interaction between killer and supporter microglia cells in vivo. Moreover, how microglia contribute to neuronal death and regeneration in vivo should be also subjected to further investigation.

It has been discussed for a long time whether activated microglia are beneficial or harmful to neurons. The challenge is to determine what sort of pathological scenarios can transform microglia into autoaggressive effector cells that attack healthy neurons and cause neuroinflammation and neurodegeneration (2, 5, 32, 35, 36, 41). We are the first to document that Irg1 selectively and functionally expressed in killer microglia in vivo (Fig. 4). Neurotoxicity of killer microglia is exerted by means of the CCL25/CCR9/Irg1 pathway (Fig. 5). Under normal and especially under pathological conditions, neuronal well being and proper functioning are highly dependent on the presence of large numbers of glial cells that sustain an abundance of neurosupportive functions. Microglial facilitation of cell death is likely to also play an important role in the elimination of superfluous cells during CNS development. In some chronic CNS diseases, such as Alzheimer’s disease, the neurodegeneration in Alzheimer’s disease is caused by autoaggressive microglia that produce neurotoxins in response to continued amyloid (Abeta) exposure (42). HIV is known to target primarily microglia in the CNS. Persistent infection of microglia with HIV can result in depleting and/or disabling microglia, leading to opportunistic CNS infections, neurodegeneration, and dementia. We have shown that microglia are capable of performing both neuroconstructive and neurodestructive functions. CCR9 and/or Irg1 siRNA significantly knock down target expressions in killer microglia and optimistically reduce mortality and change the expressive and functional profiles of killer and supporter microglia in vivo in TpTi mice (Fig. 7). The observation is indicating that CCR9 and/or Irg1 are potential therapeutic targets for gene therapy of some neurodegenerative and neuroinflammatory diseases.


   Acknowledgments
 
We thank Chen Lang, Guo Hui, Zhang Ying, Cai Guobin, and Chen Zhonghua for their constructive scientific discussions, excellent technical assistance, and animal husbandry.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The study was supported by the National Key Basic Research Program of China from the Ministry of Science and Technology of People’s Republic of China (Grants 2001CB510004 and 2001CB510008), the National Natural Science Foundation of China (Grants 39870674, 30572119, 30030130, and 30471509), a special grant from the Personnel Department of Wuhan University, China, and the Research Foundation of Health Department of Hubei Provincial Government, China (Grant 301140344). T.J. is a Chang Jiang Scholar supported by Chang Jiang Scholars Program from Ministry of Education, People’s Republic of China, and Li Ka Shing Foundation, Hong Kong, People’s Republic of China. Back

2 H.L., Z.G., H.Y., and X.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Tan Jinquan, Department of Immunology, Wuhan University School of Medicine, Dong Hu Road 115, Wuchang 430071, Wuhan, People’s Republic of China. E-mail address: jinquan_tan{at}hotmail.com Back

4 Abbreviations used in this paper: Irg1, immunoresponsive gene 1; STAg, soluble tachyzoite Ag; rLCMVNP, recombinant nucleoprotein immunodominant domain of lymphocytic choriomeningitis virus; siRNA, small interfering RNA. Back

Received for publication December 21, 2005. Accepted for publication July 4, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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