Bacterial DNA Confers Neuroprotection after Optic Nerve Injury by Suppressing CD4⁺CD25⁺ Regulatory T-Cell Activity

Abstract

purpose. Protective autoimmunity attenuates secondary degeneration after central nervous system (CNS) injury. Such neuroprotection is achieved via activation of autoimmune CD4⁺CD25⁻ effector T cells (Teffs) or suppression of naturally occurring CD4⁺CD25⁺ regulatory T cells (Tregs). In this study the ability of bacterial DNA, characterized by unmethylated CpG islands, to downregulate Treg activity and therefore, to confer neuroprotection was investigated.

methods. The effects of CpG on suppressive activity of mouse Tregs were studied by coculturing Tregs with Teffs and measuring proliferation by radiolabeled thymidine. The neuroprotective effects of CpG-mediated Treg suppression was examined in rats after optic nerve crush.

results. Teff proliferation in response to T-cell receptor stimuli was significantly reduced when the Teffs were cocultured with Tregs, compared with Teff activation when cultured alone. Treating Tregs with CpG reduced their suppressive activity and restored Teff proliferation to baseline levels. CpG injection in rats with optic nerve crush conferred significant neuroprotection compared with that in untreated control rats (118 ± 8 cells/mm² vs. 69 ± 5 cells/mm², respectively; mean ± SEM; P < 0.05). CpG-mediated neuroprotection was accompanied by significantly increased T-cell infiltration at the injury site. Similar CpG treatment of athymic nude rats yielded no neuroprotection, further suggesting a T-cell-dependent mechanism of CpG action.

conclusions. These findings strongly support the notion that alleviation of Treg suppression after injury benefits neuronal survival. Bacterial DNA attenuation of Treg suppressive activity may represent an evolutionary adaptation that curbs the amplified infection risk after CNS trauma, due to blood-brain barrier breakdown. This study may prompt development of new neuroprotective therapies aimed at the immune system, to benefit the injured CNS.

Acute trauma to the central nervous system (CNS) often results in a breakdown of the blood-brain barrier (BBB), potentially exposing the CNS to pathogenic infections. ¹ A primary insult to the CNS causes direct and immediate neuronal loss. Injury to the soma may result in the death of the cell body. Alternatively, damage to axons may result in Wallerian degeneration, in which the axonal process distal to the injury site degrades because of a lack of neurotrophic supply from the cell body. In the case of the optic nerve, death of the cell body follows soon after direct axonal injury. ^{2 3}  

Of importance, a prolonged amplification of death in neurons that escape the initial injury usually takes place and is termed secondary degeneration. ⁴ This occurs when the primary death of neurons results in leakage of intracellular neurotransmitters and reactive oxygen species, a disruption of extracellular ion balances, a general metabolic and growth factor deficit, and harmful local inflammation, including reactive gliosis. Secondary degeneration plays an important role in the pathophysiology of several neurodegenerative disorders, including glaucoma. ⁵ Induction of a “protective autoimmune” response has been shown to attenuate secondary degeneration after optic nerve injury, ^{6 7 8} after spinal cord contusion injury, ⁹ and in a model of glaucoma induced by ocular hypertension. ¹⁰ The characteristic activation and migration of CD4⁺CD25⁻ T effector (Teff) cells specific for CNS self-antigens to the site of injury can be induced by vaccination with the specific antigen ⁶ or through downregulation of naturally occurring CD4⁺CD25⁺ T regulatory (Treg) cells. ^{7 8} Modulation of Treg suppressive activity may be superior in directing vaccination for neuroprotection by reducing the likelihood of autoimmune disease development. ^{7 11 12}  

Treg cells comprise approximately 10% of the total CD4⁺ T-cell population and are thymus derived. Tregs ensure peripheral tolerance by keeping autoimmune T cells in a state of nonresponsiveness; these cells acquire an anergic phenotype and do not undergo division. ^{13 14} In addition, they do not produce interleukin (IL)-2, but are dependent on it for cell viability and function. ¹⁴ Treg-imposed suppression is a multifactorial process, involving cell-to-cell contacts ¹⁵ and the activity of soluble factors, which presumably include IL-10 ¹⁶ and TGF-β. ¹⁷ Foxp3 is a transcription factor expressed by Treg cells, which is required for their function as Tregs. Although the precise mechanism of Foxp3 action in Tregs is not known, Teff cells that are induced to express Foxp3 switch toward a regulatory phenotype. ^{18 19 20 21}  

Bacterial DNA is characterized by islands of cytosine followed by guanine (CpG) in which both bases are unmethylated (as opposed to eukaryotic DNA, in which CpG is largely methylated). Unmethylated CpG is recognized by toll-like receptor (TLR)-9, part of the TLR class of receptors that is an important part of the immune system’s microbial recognition process. Immune recognition of bacterial DNA through TLR-9 leads to the generation of a Th1 inflammatory response necessary to combat infection, but it is not known to have a direct stimulatory effect on resting T cells. ^{22 23} Recently, it was demonstrated that human Treg activity can be modulated by treatment with poly-G10-tailed oligonucleotides, which are recognized by TLR-8. ²⁴ Using synthetic oligonucleotides containing unmethylated CpG or GpC motifs and poly-G10, the investigators were able to suppress human Treg activity in vitro and then showed, in vivo, that this action could attenuate the development of cancer. 

We hypothesized that neuroprotective autoimmunity may be induced via a similar suppression of Treg activity through treatment with bacterial DNA. As such, CpG-induced suppression of Treg activity may enhance neuronal survival in the context of CNS injury. We, therefore, demonstrate an apparent evolutionary adaptation whereby immune system recognition of bacterial DNA, and the subsequent T-cell response, is beneficial to neuronal survival after optic nerve crush injury. 

Materials and Methods

Animals

Inbred female adult (8–12 weeks old) SJL/J mice and Lewis rats were purchased from Jackson Laboratories (Bar Harbor, ME), and athymic nude rats (Cr:NIH-rnu) were purchased from the National Cancer Institute (Bethesda, MD). T cells collected from mice were used for the in vitro experiments, whereas rats were used for the in vivo experiments. The animals were housed in light- and temperature-controlled rooms and matched for age in each experiment. The animals were handled according to the regulations formulated by the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska Medical Center and in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Antigens

CpG oligodeoxynucleotide (TCC-ATG-ACG-TTC-CTG-ATC-CT) and non-CpG oligodeoxynucleotide (GpC; GCT-TGA-TGA-CTC-AGC-CGG-AA) were purchased from TIB Molbiol, LLC (Aldelphia, NJ). GpC was used as negative control oligodeoxynucleotide because it does not contain the unmethylated CpG sequence recognized by TLR-9. 

Purification of Murine CD4⁺CD25⁺/CD4⁺CD25⁻ T Cells

CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were isolated as previously described. ⁸ Briefly, lymph nodes (axillary, inguinal, superficial cervical, mandibular, and mesenteric) and spleens were harvested and mashed. T cells were purified (enriched by negative selection) on CD3 cell columns (R&D Systems, Minneapolis, MN). The enriched T cells were incubated with anti-CD8 microbeads (Miltenyi Biotec, Auburn, CA), and negatively selected CD4⁺ T cells were incubated with phycoerythrin (PE)-conjugated anti-CD25 (30 μg/10⁸ cells) in phosphate-buffered saline (PBS)/2% fetal calf serum. They were then washed and incubated with anti-PE microbeads and subjected to magnetic separation (AutoMACS; Miltenyi Biotec). The retained cells were eluted from the column as purified CD4⁺CD25⁺ Treg cells. The negative fraction consisted of CD4⁺CD25⁻ Teff cells. Purified cells were cultured in 24-well plates (1 mL) with T-cell-depleted spleen cells as accessory cells (irradiated with 3000 rad) and 0.5 μg/mL anti-CD3, supplemented with 100 units of mouse recombinant (mr)IL-2. 

Activation of Treg Cells

Purified Treg cells (0.5 × 10⁶ cells/mL) were activated in RPMI medium supplemented with l-glutamine (2 mM), 2-mercaptoethanol (5 × 10⁻⁵ M), sodium pyruvate (1 mM), penicillin (100 IU/mL), streptomycin (100 μg/mL), nonessential amino acids (1 mL/100 mL), and autologous serum 2% (vol/vol) in the presence of mrIL-2 (5 ng/mL) and soluble anti-CD3 antibodies (UK1 ng/mL; Serotec, Oxford, UK). Irradiated (2500 rad) splenocytes (1.5 × 10⁶ cells/mL) were added to the culture. Cells were activated for 96 hours. For the final 24 hours of activation, CpG or GpC (2 μg/mL) or vehicle (PBS) was added to the Treg culture. 

Inhibition Assay: Coculturing of Teff and Treg Cells

The following cultures were established in 96-well flat-bottomed plates in the presence of irradiated splenocytes (10⁶/mL) supplemented with anti-CD3 antibodies (Serotec): naïve Teff cells (50 × 10³ cells/well) treated with CpG, GpC, or vehicle (PBS); activated Treg cells (50 × 10³ cells/well) treated with CpG, GpC, or vehicle; and naïve Teff and activated Treg cocultured in a 1:1 ratio treated with CpG, GpC, or vehicle. Cell populations were cultured for 72 hours. [³H]-thymidine (1 μCi) was added for the last 16 hours of culture. After the cells were harvested, their [³H]-thymidine content was analyzed by the use of a γ-counter. 

Polymerase Chain Reaction

Total RNA was purified (RNeasy Mini Kit; Qiagen Sciences, Inc., Germantown, MD). The following primers were used for Foxp3: sense, 5′-CAGCTGCCTACAGTGCCCCTAG-3′; and antisense, 5′-CATTTGCCAGCAGTGGGTAG-3′. 

Optic Nerve Crush Injury

The optic nerve was subjected to crush injury, as previously described. ³ Briefly, rats were deeply anesthetized by intramuscular injection of xylazine (XYL-M 2%, 10 mg/kg; VMD, Arendonk, Belgium) and ketamine (Ketaset, 50 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA). Temporal canthotomy was performed in the right eye, guided by a binocular operating microscope, and the conjunctiva was incised lateral to the cornea. Care was taken to preserve the retinal blood supply. After separation of the retractor bulbi muscles, the optic nerve was exposed intraorbitally by blunt dissection. With calibrated cross-action forceps, the optic nerve was subjected to a 10-second crush injury 1 to 2 mm from the eye. The contralateral nerve was not injured. 

Immunization

Immediately after optic nerve crush, the rats were injected subcutaneously with 100 μg CpG or GpC. This dosage was chosen because it has been shown to trigger a significant T-cell response in rats. ²⁵ One hundred micrograms of the oligodeoxynucleotides was dissolved in 300 μL of PBS and injected into two sites (150 μL per site) on the back of the neck. Two injections of 150 μL PBS in each rat of another group served as the negative control. 

Measurement of Secondary Degeneration by Retrograde Labeling of Retinal Ganglion Cells

Secondary degeneration of the optic nerve axons and their attached retinal ganglion cells (RGCs) was measured after postinjury application of the fluorescent lipophilic dye, 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-Asp; Invitrogen-Molecular Probes Europe BV, Leiden, The Netherlands), distal to the lesion site using a widely published method. ^{3 7 8 26 27} Briefly, rats were anesthetized, as described for optic nerve crush, 2 weeks after injury. The optic nerve was exposed again and visualized under a dissecting microscope, as previously for optic nerve crush, and the optic nerve was transected distal to the injury site, leaving the outer sheath partially intact but completely severing all the axons. A small crystal of 4-Di-10-Asp was placed in the optic nerve sheath, and approximately 100 μL of incomplete Freund’s adjuvant was applied to the area. Five days after dye application, the retinas were detached from the eye, prepared as flattened wholemounts in a 4% paraformaldehyde solution, and examined for labeled RGCs by fluorescence microscopy. ³ RGCs appeared as compact round cells that may have extended long thin processes (see 1 Fig. 2c ). Larger cells with thick, short protrusions and uneven staining were probably macrophages that had engulfed Di-Asp-labeled RGCs. These cells were observed in only a few instances and were excluded from the RGC count. It should be noted that a possibility of counting macrophages as RGCs still existed, as Di-Asp-labeled retinas were not counterstained with macrophage-microglial markers. ^{28 29} RGC counts were performed on 16 fields per retina each centered 1.5-mm from the optic nerve head. Cell counts were performed with a 40× objective (representing a 0.28-mm² area) by an investigator who was masked to the treatment group identities. At least six animals per treatment group were included in each experiment. As the assessment of RGC survival was performed 5 days after dye application, subsequent primary death of RGCs could have occurred due to optic nerve transection. Therefore, the effect of “neuroprotection” should be addressed as protection from secondary degeneration during the first 2 weeks between crush and treatment and dye application, as well as primary degeneration during the last 5 days between dye application and assessment of the retina. 

Immunohistochemistry of the Optic Nerve

At 3 days, 1 week, or 2 weeks after crush, separate groups of Lewis rats were euthanatized by carbon dioxide exposure and decapitated. These animals did not undergo the retrograde RGC labeling procedure. Optic nerves were excised, snap frozen, and stored at −20°C. Nerves were cryosectioned longitudinally into 18-μm sections, picked up directly onto gelatin-coated microscope slides, and stored at −20°C until they were stained. The slices were fixed in 95% ethanol for 10 minutes at room temperature, washed twice in 1× PBS, and incubated for 1 hour in a blocking solution consisting of 1× PBS, 0.05% Tween 20, and 20% fetal calf serum in distilled water at room temperature. Primary antibody (CD3 [clone IF4], NF-H200, NF-L70, ED1 [CD68] all from Serotec; GFAP from Abcam, Cambridge, MA) was diluted in blocking solution (CD3, 1:25; NF, 1:500; ED1, 1:100; and GFAP, 1:500) and added to the slices for 90 minutes. Slices were washed twice with 1× PBS. Secondary antibody was diluted in blocking solution (1:250) and added to slices for 60 minutes. The slides were washed twice in 1× PBS and dried. All slices were viewed in a masked fashion with a fluorescence microscope within 24 hours of staining. T cells, fluorescently labeled with anti-CD3, were visualized to be present only at the injury site when viewed with a 20× objective 3 (Fig. 4d) . However, individual cells were impossible to quantify at this magnification, and therefore T cells were counted by a masked investigator who assessed two fields per optic nerve section at the site of injury (located on either side of the crush site’s medial line) using a 40× objective (Fig. 4c) . Two fields adequately covered the site of injury and T-cell infiltration and T cells could not be found in the optic nerve outside the site of injury. At least five sections per optic nerve and three optic nerves per group were assessed. 

Statistical Analyses

T-cell proliferation was quantified with radioactive thymidine uptake and expressed as counts per minute (cpm). Each experimental group contained at least three cultures, and the results of these were averaged. Comparisons between groups were made using unpaired two-tailed t-tests. The number of RGCs were quantified in 16 separate fields per retina and averaged to determine an RGC count for each animal. The RGC counts for the animals in each experimental group were then averaged to determine the RGC count in each group. One-way ANOVAs with posttest Bonferroni adjustment were used to compare mean RGC counts between the four treatment groups of Lewis rats and unpaired, two-tailed t-tests were used to compare the two treatment groups of athymic nude rats. This RGC survival assessment was performed in Lewis rats for three separate but identical experiments, all yielding similar results, and one representative data set is presented. T cells were counted in two fields at the site of injury of a given optic nerve section, and these values were averaged to determine the T cell count per section. T-cell counts for the sections in each experimental group (at least 15 optic nerve sections from three optic nerves per group) were then averaged to determine the T-cell count for each group. One-way ANOVAs with posttest Bonferroni adjustment were used to compare mean T-cell counts between groups. 

Results

To determine whether CpG- or GpC (a negative control motif that is not recognized by TLR-9)-containing oligonucleotides can affect murine Treg activity, Treg cells were incubated with or without 2 μg/mL CpG or GpC for 24 hours and then cocultured with naïve CD4⁺ cells in the presence of anti-CD3 for 72 hours before radioactive thymidine incorporation was measured. Figure 1ademonstrates that Teff proliferation was unaffected by direct treatment with CpG or GpC. Treg cells are anergic and therefore did not proliferate when cultured in isolation. When cocultured in a 1:1 ratio, Teff proliferation was suppressed by Tregs, but this action was almost completely abrogated when CpG-treated Tregs were used. In contrast, GpC treatment of Tregs had no effect on their ability to suppress Teff proliferation (Fig. 1a) . The general phenotype of Treg cells was not affected by either CpG or GpC, as the expression level of the Foxp3 transcription factor did not change after either treatment (Fig. 1b) . 

Downregulation of Treg can translate into neuroprotection after CNS injury in vivo. ^{6 7 8} To test this effect in the current model, we crushed the optic nerves of Lewis rats with calibrated forceps and injected the animals subcutaneously with 300 μL of PBS (PBS) with or without 100 μg CpG or GpC. A group of healthy control animals received no crush injury. Two weeks later, surviving RGCs were labeled retrogradely. The retinas were then excised 5 days later, wholemounted, and analyzed under fluorescence microscopy for labeled RGCs. Optic nerve crush with PBS treatment resulted in a significant (P < 0.001) loss of RGCs (69 ± 5 cells/mm² [n = 7], mean ± SEM, Table 1 ) compared with healthy control rats (382 ± 11 cells/mm² [n = 13], Table 2 ). Treatment with CpG increased RGC survival by at least 70% when compared to PBS treatment (118 ± 8 cells/mm² [n = 6]; P < 0.05, Table 1 , Fig. 2 ), but GpC treatment did not affect neuronal survival (67 ± 3 cells/mm² [n = 6], Table 1 , Fig. 2 ). Similar results were obtained in each of three independent experiments, with one representative data set being presented. It is likely that this result is a combination of neuroprotection from both secondary and primary degeneration. Secondary degeneration occurs for the 2 weeks between the crush-treatment and dye application and primary degeneration occurs during the 5 days between dye application and retina assessment. Both of these processes could be affected by the treatment. 

In addition to an increase in surviving RGC bodies, CpG treatment led to greater immunoreactivity for heavy (200 kDa) and light (70 kDa) neurofilaments throughout the injured optic nerve of Lewis rats. This indicates an increased preservation of RGC axons distal to, within, and proximal to the injury site (Fig. 3a) . Injured optic nerve sections were also immunolabeled for GFAP (an astrocyte marker) and ED1 (which labels macrophages and microglia). Immunohistochemistry demonstrated the presence of microglia within the injury site and astrocytes surrounding the injury site. ED1-positive staining represents microglial populations resident in the optic nerve, but can also label infiltrating blood-borne macrophages. There did not appear to be a difference in astrocytic or microglial response to injury due to treatment with CpG or GpC at any time points tested (Fig. 3b) . 

Immunolabeling for CD3 in the optic nerve, 3 days after crush, indicated a significant (P < 0.001) upregulation of the T-cell response at the site of injury in rats treated with CpG (69 ± 5 cells/mm²) compared with PBS (34 ± 4 cells/mm²). However, GpC (38 ± 3 cells/mm²) did not produce any such effect, suggesting an association between T cell infiltration and neuroprotection (Fig. 4a 4b) . No T-cell infiltrates were found in the contralateral (noninjured) optic nerves or outside the injury site (data not shown). The T cells were counted under high magnification (40×), and representative fields are shown (Fig. 4c) . T cells were localized to the site of the injury (Fig. 4d) . 

To further substantiate a T-cell-mediated mechanism of action, we repeated the experiment in athymic nude rats, which are devoid of T cells. In this context, CpG had no neuroprotective effect compared with PBS (36 ± 7 cells/mm² [n = 6] vs. 58 ± 13 cells/mm² [n = 6], respectively; Figs. 5a 5b ). This level of survival is comparable to, but slightly lower than, the RGC survival in Lewis rats. Of interest, neuronal survival appeared to be slightly (though not significantly) impaired in the group of athymic rats treated with CpG. The trend toward exacerbation of neuronal loss in the CpG-treated rats might have been the result of a robust innate immune response to the bacterial DNA; this may have resulted in detrimental inflammation at the site of injury in the absence of beneficial T-cell-mediated neuroprotection in these athymic rats. 

Discussion

Treatment with bacterial DNA attenuated secondary degeneration after optic nerve injury via downregulation of Treg suppressive activity. Injection of CpG into wild-type rats resulted in a significant increase in survival of RGCs and an increase in immunoreactivity for neurofilament throughout the optic nerve. This neuroprotective effect was accompanied by a localized accumulation of T cells at the injured site. No benefit was observed with CpG treatment of immune-deficient rats, further suggesting that the neuroprotective effect is mediated by T cells. 

Our findings are consistent with previously published results that a controlled boost of T-cell immunity attenuates the spread of secondary neuronal damage and thus leads to a better outcome. ^{6 7 8 31 32 33 34 35 36} The approach of Treg suppression as a means of autoimmune response induction is safer than a direct boost of autoimmune T cells; the Treg cells after treatment with CpG did not lose the suppressive phenotype, as evidenced by the lack of change in Foxp3 expression. Therefore, it is possible that some level of peripheral tolerance was maintained or could have been therapeutically reinstated. However, Treg suppressive activity was attenuated by CpG, thereby allowing activation of T cells, which led to neuroprotection in the CNS. 

It should be noted that we assessed infiltration of CD3⁺ cells at the injury site, but did not distinguish between autoimmune Teff and Treg cells. Previous studies demonstrate that T cells, which infiltrate the injury site after CNS lesion, and protective autoimmunity induction are specific to self-antigen located at the injury site. ^{37 38} Further work, however, is needed, to characterize the antigenic specificity of these infiltrating T lymphocytes. 

The importance of T cells in the beneficial effect of CpG is evidenced by in vitro assays measuring T-cell proliferation in the presence of CpG-treated Tregs and in vivo studies showing T-cell infiltration into the optic nerve in rats after CpG treatment. In addition, CpG did not induce neuroprotection in nude athymic rats. The lack of neuroprotection in nude rats should be received with some degree of caution, since no wild-type rats with a background identical with the Cr:NIH-rnu athymic rats that were used in this study exist for control experiments. Future studies involving immune-deficient mice with adequate controls as well as TLR-9 knockout mice will be used to investigate further the role of T cells in CpG-mediated neuroprotection. The present study found no changes in astroglial or microglial responses at the site of injury in CpG-treated animals compared with control or GpC-treated rats. There is still a possibility that the effect of CpG is mediated, at least in part, through oligodendrocytes or directly on neurons. This will be examined in future studies, in primary glial and neuronal cultures. 

Recent work has shown similar results with another compound, dopamine, which specifically interacts with Treg cells and results in suppression of their regulatory activity. ⁸ Type I dopamine receptors are preferentially expressed by Tregs. ⁸ On interaction with Tregs via these receptors, dopamine induced a downregulation of Erk1/2 activation, leading to reduced production of anti-inflammatory-suppressive cytokines, thereby alleviating the suppressive activity of Tregs. ⁸ The mechanism underlying CpG-induced suppression of Treg inhibitory effects is not understood and is currently under investigation. Of interest, treatment with GpC did not affect the suppressive properties of murine Tregs; this suggests that unlike human Tregs cells, ²⁴ murine Treg cells respond to bacterial DNA by TLR-9 recognition of CpG rather than by TLR-8 recognition of poly-G10. 

It should be noted that a recent study ³⁰ has suggested that a ketamine-xylazine anesthetic regimen may be neuroprotective after optic nerve transection. In the context of the present study, it may be that the anesthetic compounds improved RGC survival to some extent. Since all the animals in the present study received the same anesthetic treatment; however, we attribute the neuroprotective effect in the treatment groups to the action of injected CpG. Any potential neuroprotective effect that may have been masked by the effect of anesthesia on RGC survival cannot be assessed at present. 

Suppression of Tregs by bacterial antigens can be viewed as an evolutionary adaptation of the immune system. Bacterial antigens may serve as stress-damage signals ³⁹ and thus lead to attenuation of Treg-suppressive activity, eliciting the strongest immune response against invaders. However, the surprising finding that the natural immune response to bacterial antigens can be beneficial after CNS insult remains consistent with previously published data showing a beneficial effect of Treg depletion on neuronal survival. ^{6 7 8} As infection often occurs after acute CNS trauma and BBB breakdown, ³⁹ the current results may represent an adaptation process that provides a mechanism (downregulation of Tregs) for an efficient antibacterial immune response after injury. This mechanism also may lead to a beneficial neuroprotective immunity and attenuation of neuronal loss. Our results suggest that clinical trials may be useful for further evaluation of these properties, which could lead to the development of a new generation of therapeutic interventions for neuroprotection to target the immune system rather than the damaged neurons directly. 

³  Present affiliation: Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom.

 Supported in part by the Nebraska Tobacco Settlement Biomedical Research Development Fund and Pilot Award Grant PP1221 from the National Multiple Sclerosis Society (JK); an unrestricted grant from Research to Prevent Blindness (CBC); a Summer Student’s Fellowship from Fight For Sight; and a Gates Cambridge Scholarship (TVJ).

 Submitted for publication November 9, 2006; revised February 12, March 7, and April 16, 2007; accepted May 23, 2007.

 Disclosure: T.V. Johnson, None; C.B. Camras, None; J. Kipnis, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

(a) Proliferation of Teff and/or Treg cells isolated from SJL/J mice was assayed by incorporation of [³H]-thymidine into Teffs or Tregs cultured in isolation or together. Recorded results are from one representative experiment of three with similar data and are expressed as the mean ± SD of four replicates. Tregs were activated by incubation for 96 hours with anti-CD3 antibodies in the presence of mouse recombinant interleukin (mr)IL-2. During the last 24 hours of incubation, CpG or GpC was added to Tregs. Control Tregs were left untreated. Activation of Teffs (without Tregs) in the presence of CpG or GpC did not affect their proliferation (first set of three bars). Treg are anergic and thus did not proliferate when cultured in isolation (middle set of three bars). Activation of Treg for 72 hours, followed by the addition of CpG for the last 24 hours of activation, totally eliminated (***P < 0.008) the suppressive activity of Tregs on Teffs and led to proliferation of Teffs in a 1:1 Treg-Teff coculture (last set of three bars). Untreated Tregs completely inhibited proliferation of Teffs in coculture, and GpC did not affect the Treg-induced inhibition. (b) Polymerase chain reaction (PCR) products for Foxp3 expression in Treg after indicated treatments. β-Actin is a housekeeping gene and was used as an internal standard control.

Figure 2.

 

In Lewis rats under heavy anesthesia, the right optic nerves were crushed with calibrated forceps, immediately followed by subcutaneous injection of 300 μL of PBS, with or without 100 μg CpG or GpC. After 2 weeks, RGCs were labeled and counted. (a) Bars represent the mean RGC count per square millimeter of each group. Error bars represent the SEM. Injection of CpG resulted in significant (*P < 0.05) neuroprotection compared with GpC or PBS. (b) Each dot in the dot plot represents the average number of cells per square millimeter counted in 16 fields in a single retina. (c) Representative micrographs of the retinal fields.

Figure 3.

 

(a) Longitudinal sections of injured optic nerves of Lewis rats were immunolabeled for light (70 kDa) and heavy (200 kDa) neurofilaments and analyzed under confocal microscopy. Fields were analyzed at the site of injury as well as at areas distal to and proximal to the injury site. Treatment with CpG was associated with an increase in immunoreactivity of neurofilaments in all three areas compared with PBS treatment, indicating an increased survival of RGC axons. At least three animals per group were tested, all yielding similar results. Representative micrographs are shown. (b) Longitudinal sections of injured optic nerves were immunolabeled for ED1 (red) and GFAP (green) and analyzed using fluorescence microscopy. Staining patterns demonstrate a homing of microglia to the site of injury and a fleeing of astrocytes from the injury site. There does not appear to be an effect of CpG or GpC treatment on the microglial or astrocytic response to CNS injury.

Figure 4.

 

Longitudinal sections of injured optic nerves (3 days after crush) were immunolabeled for CD3 and analyzed under fluorescence microscopy. At least three animals per group and five tissue sections per animal were tested, all yielding similar results. (a) Bars represent the mean CD3⁺ cell count per square millimeter of each group. Error bars, the SEM. A significant (***P < 0.001) increase in T-cell accumulation was evident in Lewis rats treated with CpG, as opposed to treatment with PBS or GpC. (b) Each dot in the dot plot represents the average T cell count in a single optic nerve section. (c) Representative micrographs of counted fields are shown. (d) A representative micrograph at lower magnification shows that T cells were localized to the site of injury.

Table 1.

 

Retinal Ganglion Cell Counts in Control, CpG-, and GpC-Treated Rats after Optic Nerve Crush

Treatment	Animal	RGCs in Each Field (n)															Average Cells/Field	Average Cells/mm2
PBS	1	21	24	19	18	16	16	18	17	19.7	69.6
		10	19	25	29	16	25	22	20
	2	2	35	20	24	23	22	21	21	22.2	78.5
		19	10	25	26	20	14	45	28
	3	4	10	25	24	23	29	19	18	20.7	73.2
		10	42	15	32	18	28	19	15
	4	17	21	25	2	25	25	26	30	20.4	72.3
		20	10	12	11	25	32	26	20
	5	14	10	10	15	16	8	7	10	12.3	43.6
		21	12	5	12	5	15	19	18
	6	19	36	20	3	23	13	16	28	24.1	85.3
		21	41	27	30	29	25	40	15
	7	10	9	10	48	29	24	12	13	17.8	63.0
		14	18	24	16	5	21	10	22
CpG	1	39	42	29	46	40	15	36	35	39.4	139.5
		41	52	26	62	48	26	52	42
	2	25	45	31	7	32	30	32	30	33.4	118.3
		52	23	26	25	29	56	51	41
	3	30	36	29	45	12	54	31	60	37.1	131.3
		42	16	36	54	40	47	26	36
	4	46	29	48	42	32	28	42	25	35.4	125.1
		35	26	29	52	45	23	38	26
	5	19	5	32	26	10	17	15	5	25.1	88.7
		25	32	36	30	29	20	40	60
	6	30	36	45	26	25	28	42	32	29.8	105.2
		7	52	39	34	12	25	24	19
GpC	1	19	23	24	19	12	19	20	15	18.8	66.5
		15	12	20	23	22	15	20	23
	2	15	13	19	20	25	23	29	20	17.8	62.8
		23	10	11	12	23	20	16	5
	3	32	26	10	12	23	22	12	20	20.6	73.0
		25	12	29	27	27	24	10	19
	4	13	29	36	29	19	23	12	15	22.1	78.3
		15	16	42	15	13	16	29	32
	5	16	15	25	26	24	28	15	20	17.8	62.8
		10	10	13	16	25	16	10	15
	6	10	12	16	23	12	10	14	15	16.6	58.6
		24	21	23	20	12	19	17	17

 In adult Lewis rats under heavy anesthesia, the right optic nerves were crushed with calibrated forceps, immediately followed by subcutaneous injection of 300 μL of PBS, with or without 100 μg CpG. After 2 weeks, RGCs were labeled and counted in 16 fields equidistant from the optic nerve at 40× magnification (each field covered 0.28 mm²). Treatment with CpG resulted in a significantly (P < 0.05) greater number of surviving RGCs compared with PBS or GpC. The data represent one representative experiment of three independent experiments.

Table 2.

 

RGC Density in Healthy Lewis Rats

	Animal																									Avg.
		1	2	3	4	5	6	7	8	9	10	11	12	13
RGCs (n/mm2)	382	410	383	313	365	372	432	398	444	432	322	349	364	382

 Retinal ganglion cells were labeled in healthy Lewis rats and assessed for baseline cell density. The cells were counted in 16 fields equidistant from the optic nerve at 40× magnification (each field covered 0.28 mm²) and averaged to determine the RGC density in each animal.

Figure 5.

 

(a) In adult athymic nude rats under heavy anesthesia, the right optic nerves were crushed with calibrated forceps, immediately followed by subcutaneous injection of 300 μL of PBS, with or without 100 μg CpG. After 2 weeks, RGCs were labeled and counted. (a) Bars represent the mean RGC count per square millimeter of each group. Error bars, SEM. Athymic nude rats treated with CpG did not demonstrate any neuroprotection compared with PBS-treated counterparts. (b) Each dot in the dot plot represents the average cell/per square millimeter in a single retina. (c) Representative micrographs of retinal fields are shown.

The authors thank Ashley Reynolds for assistance with inhibition coculture assays.