Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

Abstract

CD200 has been reported to be neuroprotective in neurodegenerative diseases. However, the potential protective effects of CD200 in germinal matrix hemorrhage (GMH) have not been investigated. We examined the anti-inflammatory mechanisms of CD200 after GMH. A total of 167 seven-day-old rat pups were used. The time-dependent effect of GMH on the levels of CD200 and CD200 Receptor 1 (CD200R1) was evaluated by western blot. CD200R1 was localized by immunohistochemistry. The short-term (24 h) and long-term (28 days) outcomes were evaluated after CD200 fusion protein (CD200Fc) treatment by neurobehavioral assessment. CD200 small interfering RNA (siRNA) and downstream of tyrosine kinase 1 (Dok1) siRNA were injected intracerebroventricularly. Western blot was employed to study the mechanisms of CD200 and CD200R1. GMH induced significant developmental delay and caused impairment in both cognitive and motor functions in rat pups. CD200Fc ameliorated GMH-induced damage. CD200Fc increased expression of Dok1 and decreased IL-1beta and TNF-alpha levels. CD200R1 siRNA and Dok1 siRNA abolished the beneficial effects of CD200Fc, as demonstrated by enhanced expression levels of IL-1beta and TNF-alpha. CD200Fc inhibited GMH-induced inflammation and this effect may be mediated by CD200R1/Dok1 pathway. Thus, CD200Fc may serve as a potential treatment to ameliorate brain injury for GMH patients.

Introduction

Germinal matrix hemorrhage (GMH) is defined as the rupture of immature blood vessels within the subependymal brain tissues and is the most common neurological disorder of newborns.¹ It occurs approximately 3.5 times per 1000 live births.² This is an important clinical problem. However, current clinical management is limited. More research is needed to investigate innovative therapeutic modalities. Debilitating consequences of GMH include neurological deficits, epilepsy, and the formation of post-hemorrhagic hydrocephalus.^1,2 Mounting evidence shows that microglia mediate neuroinflammation contributing to secondary brain injuries after GMH.^3–5 Therefore, ameliorating inflammation can be a therapeutic strategy for GMH.

CD200 and its receptor, CD200 Receptor (CD200R), have unique roles in reducing inflammatory processes. CD200 binds to CD200R and triggers an anti-inflammatory signaling cascade in CD200-expressing cells.^6–8 CD200 is a member of the immunoglobulin superfamily.^9,10 Its expression level is relatively high in the brain, particularly on neurons.^6,11 CD200R is highly glycosylated with a DNA sequence coding for a protein with homology to CD200 and is expressed on perivascular macrophages and microglia.¹² In experimental animal models, impairment of CD200–CD200R1 interaction resulted in severe pathology.^13–15 On the contrary, enhancement of CD200R1 signaling alleviated pathological outcomes.^16–18 Particularly, inflammatory responses were attenuated by a CD200 fusion protein (CD200Fc) that stimulates CD200R activation.^19,20 Downstream of tyrosine kinase 1 (Dok1) belongs to the downstream key signaling protein of CD200R1. CD200–CD200R1 interaction resulted in inducing Dok1.^21,22 Direct interaction of the adaptor protein Downstream of tyrosine kinase 2 (Dok2) with a CD200R cytoplasmic domain (Y345) results in the binding and activation of RasGAP which inhibits Ras signaling pathways and consequently upregulates downstream inflammatory signals. However, CD200–CD200R1 interaction induces adaptor protein Dok1, which binds to another phosphotyrosine residue (Y362) on CD200R and reverses Dok2–RasGAP inhibition of Ras signaling (Supplementary Figure 1). In this study, we will focus on the role of Dok1 in this signaling pathway.

CD200–CD200R1 signaling has been shown to suppress microglia activation in Alzheimer's disease and Parkinson's disease.^19,23 Lack of CD200 also results in rapid onset of experimental autoimmune encephalomyelitis.²⁴ In addition, this signaling has also been evaluated in brain infection models.^22,25 However, the anti-inflammatory effect of CD200–CD200R in brain hemorrhage has never been studied before. Since the inflammatory response after brain hemorrhage can be largely different from chronic inflammation or infection, the potential benefit effects of CD200Fc in GMH are worth further characterization and investigation.

In the present study, we use unilateral collagenase infusion into the germinal matrix in neonatal rats, which mimics clinical GMH, and tested if CD200Fc treatment ameliorates inflammation, preserves blood–brain barrier (BBB) integrity and improves long-term neurobehavioral function after GMH. We also postulate that the inhibition of the CD200 pathway reverses these beneficial effects.

Materials and methods

All experiments were conducted in compliance with the NIH Guidelines for the Use of Animals in Neuroscience Research. All experiments were approved by the Loma Linda University Institutional Animal Care and Use Committee. All experiments are reported in compliance with the Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines. Neither collagenase-induced GMH nor administration of CD200Fc caused mortality in this study. Animals were numbered and randomly assigned to different experimental groups. Specifically, numbers were randomized by Excel to form different groups according to the experimental design and animals were assigned to these groups. Investigators were blinded to the experimental groups when performing neurological tests, immunofluorescence, and Evans Blue Assay. The investigators quantitating western blot densitometry and Evans Blue results were also blinded to the experimental groups.

GMH model and experimental protocol

GMH was induced as described.²⁶ For GMH induction, animals were anesthetized with 3% isoflurane and placed onto stereotaxic frame. Isoflurane concentration was then reduced to 2%. The scalp area was sterilized, and Bregma was exposed. Using Bregma as a reference point, the following stereotactic coordinates were measured: 1.6 (rostral) and 1.5 mm (lateral, right). A burr hole (1 mm) was drilled. A 27-gauge needle was inserted at a rate of 1 mm/min to a depth of 2.7 mm from the dura, where the germinal matrix is located. Using a microinfusion pump (Harvard Apparatus, Holliston, MA, USA), 0.3 U of clostridial collagenase VII-S (Sigma, St Louis, MO, USA) in 3 μl was infused through the Hamilton syringe at the rate of 1 μl/min. The needle remained in place for an additional 10 min after injection to prevent back leakage. After the needle was removed, the burr hole was sealed with bone wax and the incision was sutured. Pups were allowed to recover on a 37℃ heated blanket. On recovering from anesthesia, the animals were returned to their dams. Sham operation consisted of needle insertion alone without collagenase infusion.

Intracerebroventricular drug administration was performed as previously described.²⁷ Briefly, rats were placed in a stereotaxic apparatus under 2.5% isoflurane anesthesia. The scalp area was sterilized, and Bregma was exposed. Using Bregma as a reference point, the following stereotactic coordinates were measured: 1.0 (rostral) and 1.0 mm (lateral). A burr hole (1 mm) was drilled. A 27-gauge needle was inserted at a rate of 1 mm/min at the depth of 1.8 mm from the dura, where the lateral ventricle is located.

With a microinfusion pump (Harvard Apparatus), CD200Fc (MyBioSource, USA) in 2.0 μl (0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg) was infused at 1.0 μl/min through the Hamilton syringe at 3 h after GMH induction into the ipsilateral (right) lateral ventricle.^19,28,29 For long-term studies, the same amount of CD200Fc was injected daily for three days starting at 3 h after GMH in anesthetized animals. Two microliters of PBS (pH 7.4) were injected intracerebroventricularly (i.c.v.) to the same stereotaxic location as the vehicle treatment.

CD200R1 small interfering RNA (siRNA, 500 pmol, MyBioSource), scrambled siRNA (500 pmol, MyBioSource), and Dok1 siRNA (500 pmol, MyBioSource) in 0.5 μl PBS was infused at 1.0 μl/min through the Hamilton syringe at 24 h prior to GMH induction. The infusion method was the same as that in CD200Fc administration except that siRNA was injected into the contralateral (left) lateral ventricle.

Pregnant Sprague-Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN, USA). One hundred sixty-seven P7 rat pups of both genders were used. The sample size to be estimated for all groups with formula: sample size n = Z × Z [P(1 − P)/(D × D)]. P = Expected Frequency Value = 80%. Z = 1.960 with confidence level of 95%. D = (Expected Frequency − Worst Acceptable) = 0.25. Sample size estimates were then made using data from previous experiments. Based on the previous assumptions, the mean values, standard deviation, and up to a 20% change in means from these previous studies dictate that animals are to be used per group. All rats were conducted to corresponding surgeries and following assessments according to the experimental design (Supplementary Figure 2).

Experiment I

To determine the time course of endogenous CD200 and CD200R1 after GMH, western blot was performed to measure their expression levels. A piece of brain tissue with the width of 0.32 inch centered at the collagenase injection point was defined as the peri-hemorrhage tissue in the ipsilateral (right) hemisphere and they were collected at 0 (Sham), 3, 6, 12, and 24 h, 3 and 7 days after GMH. Forty-two rats were numbered and randomly divided using Excel into Sham group (n = 6) and GMH group (n = 36).

Experiment II

To assess the effects of exogenous CD200Fc at different dosages after GMH, body righting and negative geotaxis tests were performed at 24 h after the surgery. These two neurobehavioral tests were used to evaluate the developmental profiles particularly in neonates. However, they are not appropriate methods to assess adults' neurological function since they would be too early in adult stroke models. Fifty rats were randomly divided into five groups: Sham group (n = 10), GMH + Vehicle group (n = 10), GMH + CD200Fc (0.5 mg/kg, i.c.v.) (n = 10), GMH + CD200Fc (1.0 mg/kg, i.c.v.) (n = 10), and GMH + CD200Fc (1.5 mg/kg, i.c.v.) (n = 10). Evans Blue extravasation assay was performed at 24 h after neurobehavioral tests (n = 6).

Experiment III

To assess the inflammation reaction at 24 h after GMH and determine the expression characteristics and localization of CD200R1 at 24 h after GMH, immunofluorescence labeling was performed. Nine rats were randomly divided into Sham group (n = 3), GMH + Vehicle group (n = 3), and GMH + CD200Fc (1.5 mg/kg, i.c.v.) group (n = 3).

Experiment IV

To assess the long-term effects of i.c.v. administration of exogenous CD200Fc after GMH. Neurobehavioral tests were performed at 28 days after the surgery. Thirty rats were divided into Sham group (n = 10), GMH + Vehicle group (n = 10), and GMH + CD200Fc (1. 5 mg/kg, i.c.v., n = 10).

Experiment V

CD200R1 siRNA and Dok1 siRNA specifically knock down CD200R1 and Dok1, respectively. Intracerebroventricular siRNA administration was performed to further verify the essential role of CD200R1 signaling in the context of GMH. The expression levels of CD200, CD200R1, Dok1, IL-1beta, and TNF-alpha were evaluated by western blot. Thirty-six rats were randomly divided into Sham group (n = 6), GMH + Vehicle group (n = 6), GMH + CD200Fc (1.5 mg/kg, n = 6), GMH + CD200Fc + scrambled siRNA group (n = 6), GMH + CD200Fc + CD200R1 siRNA group (n = 6) and GMH + CD200Fc + Dok1 siRNA group (n = 6).

Neurological examination

The protective effects of CD200Fc on the development of animals after GMH were evaluated using body righting and negative geotaxis tests. Tests were performed daily in a blinded fashion at 24 h after GMH induction.²⁶ Body righting test is used to record the time it takes the rats to turn on all four limbs from a back position that they were initially placed on. Negative geotaxis test is performed by placing the rats on a 45 ° incline with head down and recording the time it takes for them to make 90 ° turn and 180 ° turn.

We also tested the protective effects of CD200Fc treatment on cognitive function using water maze, and motor function using foot fault and rotarod in a blinded fashion on Day 28 after GMH.²⁶ Foot Fault is to the animal's motor abilities. Animals will be placed on a horizontal grid floor (square size 28 × 3 cm, wire diameter 0.4 cm) for 2 min. Foot fault is defined as when the animal cannot coordinate its movements and it falls through one of the openings on the grid. The numbers of foot faults were recorded per limb for each animal using a video device. Rotarod is to test the animal's ability to remain on an accelerating rod. Animals were placed on an accelerating rod and the time it took for them to fall was recorded.

Evans Blue extravasation assay

Evans Blue extravasation assays were conducted at 24 h after the surgery as previously reported.^30,31 Briefly, pups received an intraperitoneal injection of 4.0 ml/kg Evans blue dye (4%), 3 h prior to sacrifice. Following that, deeply anesthetized animals were transcardially perfused with ice-cold PBS (50 ml) and brains were removed, divided into right and left hemispheres, snap-frozen in liquid nitrogen, and stored at −80℃. Left hemispheres were then homogenized in 500 μl PBS, sonicated and then centrifuged for 30 min with a relative centrifugal force (rcf) of 15,000. An equal amount of trichloroacetic acid (50%) was added to 500 μl supernatant and allowed to incubate overnight at 4℃ before being re-centrifuged (30 min, 15,000 rcf). The quantity of extravasated Evans blue dye was detected by spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 610 nm and quantified according to a standard curve. These data were calculated as milligrams of Evans blue dye per gram of brain tissue.

Western blots

The effects of GMH on CD200 and CD200R1 levels were evaluated at 0 (Sham), 3, 6, 12, and 24 h, 3 and 7 days after GMH by western blot as previously described.³² The effects of CD200Fc on the GMH-induced activation of the CD200 pathway were evaluated by western blot at 24 h after GMH. Primary antibodies used were CD200 (Santa Cruz Biotechnology), CD200R1 (Santa Cruz Biotechnology), Dok1 (Santa Cruz Biotechnology), IL-1beta (Abcam), and TNF-alpha (Abcam).

Immunohistochemistry and brain injury evaluation

Animals were euthanized at 24 h post-GMH and brains were processed. Ten micrometers-thick coronal sections containing the bilateral basal cerebral cortex were cut on a cryostat (Leica Microsystems, Bannockburn, IL, USA). To assess the inflammation reaction at 24 h after GMH, single staining with fluorescence labeling was performed. Sections were incubated overnight at 4℃ with mouse anti-Iba-1 (1:500, Abcam) and the appropriate fluorescence dye-conjugated secondary antibody (1:1000, Jackson Immunoresearch, West Grove, PA, USA) was applied in the dark for 1 h at 21℃. The sections were visualized with a fluorescence microscope, and the photomicrographs were saved and merged with Image Pro Plus software (Olympus, Melville, NY, USA). To determine the expression and localization of CD200R1 at 24 h after GMH, double fluorescence labeling was performed as previously described.^33,34 Sections were incubated overnight at 4℃ with goat anti-CD200R1 (1:100, Santa Cruz Biotechnology), mouse anti-Iba-1 (1:500, Abcam) according to the manufacturer's instructions. Appropriate fluorescence dye-conjugated secondary antibodies were applied in the dark for 1 h at 21℃. For negative controls, the primary antibodies were omitted and the same staining procedures were performed.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 5 (GraphPad software). Values are expressed as mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey post hoc test. A P value of < 0.05 was considered statistically significant.

Result

Endogenous CD200 and CD200R1 were downregulated after GMH

Western blot results showed that both CD200 and CD200R1 expression levels decreased at 3 h and reached the lowest level at 24 h after GMH. However, expression levels of both proteins gradually tended to recover at Day 7 (Figure 1(a) and (b)).

Figure 1.

Expression time course of CD200 and CD200R1 after GMH. (a) CD200 level decreased at 3 h, with the lowest level being at 24 h, and slowly recovered thereafter to Day 7 (*P < 0.05, n = 6 each group/time point). (b) Similarly, CD200R1 level decreased at 3 h, with the lowest level being at 24 h, and slowly recovered thereafter to Day 7 (*P < 0.05 vs Sham; n = 6 each group/time point). Values are expressed as mean ± SD.

Immunostaining of Iba-1 (marker for microglia) showed that there were more Iba-1 positive cells in the GMH group than in the Sham group. We also identified activated microglia that demonstrated different morphology from resting microglia (Figure 2(a)). Double immunostaining of CD200R1 with Iba-1 further verified that this receptor is expressed on microglia (Figure 2(b)). All immunostaining samples were collected at 24 h after GMH.

Figure 2.

Immunohistochemistry staining of Iba-1 (marker for microglia) and CD200R1 on microglia. (a) Immunostaining of Iba-1 in Sham and Vehicle-treated animals (24 h after GMH). (b) CD200R1 was expressed on microglia cells in Sham, Vehicle-treated and CD200Fc-treated groups at 24 h after GMH. (n = 3 each group, Scale bar = 30 μm).

CD200FC preserved BBB integrity, decreased inflammation, and improved neurobehavioral outcomes at 24 h after GMH

Three dosages of CD200Fc (0.5 mg, 1.0 mg, and 1.5 mg/kg) were administrated i.c.v. 3 h after GMH. BBB permeability increased after GMH. Both high and middle dosages of CD200Fc preserved BBB integrity at 24 h (Figure 3(a)). In addition, high dosage improved the neurological function at 24 h after GMH, as evaluated by both body righting and negative geotaxis tests (Figure 3(b) and (c)). Western blots showed that GMH increased the expression of IL-1beta and decreased the expression of ZO-1. High dosages of CD200Fc decreased the expression of IL-1beta. Middle and high dosages of CD200Fc also increased expression of ZO-1 at 24 h after GMH (Figure 3(d)). These results showed that GMH elicited inflammatory response and compromised BBB integrity and that CD200Fc conferred beneficial effects in ameliorating inflammation, preserving BBB integrity and improving neurological deficits.

Figure 3.

External CD200Fc (1.5 mg/kg) treatment improved BBB integrity and neurological function at 24 h after GMH. (a) GMH increased BBB permeability, as demonstrated by more Evans blue extravasation into the brain tissues and CD200Fc (1.0–1.5 mg/kg) decreased the BBB permeability in ipsilateral hemisphere at 24 h after GMH. These data were calculated as milligrams of Evans blue dye per grams of tissue. *P < 0.05 vs Sham, ^#P < 0.05 vs GMH + Vehicle, n = 6/group, one-way ANOVA, Tukey's test). GMH caused neurological deficits evaluated by body righting (b) and negative geotaxis tests (c), compared with Sham group and CD200Fc (1.5 mg/kg) improved neurological function (*P < 0.05 vs Sham, ^#P < 0.05 vs GMH + Vehicle, one-way ANOVA, Tukey's test, n = 10/group). (d) Representative western blot bands and quantitative analysis of IL-1beta and ZO-1 at 24 h after GMH. GMH increased the expression of IL-1beta and decreased the expression of ZO-1 while CD200Fc (1.5 mg/kg) attenuated these detrimental effects of GMH (*P < 0.05 vs Sham, ^#P < 0.05, vs GMH + Vehicle, n = 6/group, one-way ANOVA followed by the Tukey test). Values are expressed as a mean ± SD.

Since high dosage of CD200Fc was the most effective dosage in abovementioned studies, this dose was used for the following long-term and mechanistic studies.

CD200Fc (1.5 mg/kg) improved long-term neurological functional outcomes at four weeks after GMH

Vehicle-treated GMH animals demonstrated significant spatial memory loss compared with Sham-operated animals in the Morris water maze by swimming greater distances finding the platform (P < 0.05) and spending less time in the target quadrant during the probe trials (P < 0.05). CD200Fc-treated animals showed significant cognitive functional improvement by having reduced swimming distances (P < 0.05; Figure 4(a)) and tended to spend more time in the target quadrant during the probe trials (P > 0.05 vs Sham; Figure 4(b)). Furthermore, vehicle-treated animals had significantly more foot faults than Sham, but CD200Fc-treated animals had significantly reduced foot faults compared with the vehicle group (P < 0.05; Figure 4(c)). Vehicle animals had significantly worse rotarod performance compared with Sham, but CD200Fc treated animals had significantly better rotarod performance than vehicle rats (P < 0.05; Figure 4(d)).

Figure 4.

External CD200Fc (1.5 mg/kg) improved memory and motor functions at 28 days after GMH. (a) Swimming distance. (b) Percentage of time in the target quadrant. (c) Foot fault test. (d) Rotarod test. Overall, GMH caused deficits in all tests and CD200Fc at 1.5 mg/kg improved neurological functions. Data are presented as mean ± SD, *P < 0.05 vs Sham, ^#P < 0.05 vs vehicle, n = 10/group.

In our study, we used P7 rats to perform the surgery. At P7, it is very difficult to distinguish different genders. At P14, we distinguished different genders and separated them into different cages. Therefore, we performed a gender-based analysis for long-term cognitive dysfunctions. Both male and female results showed the exogenous CD200Fc (1.5 mg/kg) improved memory and motor functions at 28 days after GMH. There was no significant gender difference in response to the treatment (Supplementary Figures 3 and 4).

Effect of CD200R1 siRNA and DOK1 siRNA on CD200Fc treatment

In our postulation, the activation of CD200/CD200R1 signaling plays an anti-inflammatory role after the hemorrhage and this effect is mediated by Dok1. To investigate the underlying mechanisms of recombinant CD200Fc, we administrated CD200R1 siRNA along with CD200Fc treatment. After GMH, CD200Fc enhanced the expression levels of CD200, CD200R1, and Dok1. CD200R1 siRNA decreased CD200R1 protein expression. In addition, we found that the expression of Dok1 also decreased with the administration of CD200R1 siRNA (Figure 5(a) to (c)). Dok1 siRNA decreased the expression of Dok1 protein but not CD200 and CD200R1 (Figure 5(d)).

Figure 5.

The adverse effects of silencing endogenous CD200R1 or Dok1 by CD200R1 siRNA or Dok1 siRNA at 24 h after GMH. (a) Representative western blot bands. (b) Quantitative analysis of CD200 showed that protein level of CD200 increased following the treatment with CD200Fc. (c) Quantitative analysis of CD200R1 showed CD200R1 increased in the treatment group and decreased in GMH group. CD200R1 siRNA intervention significantly decreased CD200R1 level. However, CD200R1 level does not change with Dok1 siRNA intervention. (d) Quantitative analysis of Dok1 showed that Dok1 expression significantly decreased by both Dok1 siRNA and CD200R1 siRNA intervention. Data are expressed as mean ± SD, *P < 0.05 vs Sham, ^#P < 0.05 vs vehicle, ^&P < 0.05 vs Treatment, n = 6/group, one-way ANOVA followed by the Tukey test.

Knockdown CD200R1 and Dok1 enhanced inflammation factors at 24 h after GMH

GMH enhanced the expressions of IL-1beta and TNF-alpha (Figure 6(a)). CD200Fc decreased the expressions of IL-1beta and TNF-alpha. CD200R1 siRNA and Dok1 siRNA restored the expression of IL-1beta (Figure 6(b)) and TNF-alpha even in the presence of CD200Fc (Figure 6(c)).

Figure 6.

Effects of CD200R1 siRNA and Dok1 siRNA on IL-1beta and TNF-alpha expression in the presence of CD200Fc (1.5 mg/kg) at 24 h after GMH. (a) Representative western blots bands. (b) Quantitative analysis of IL-1beta. GMH enhanced while CD200Fc reduced the protein level of IL-1beta. (c) Quantitative analysis of TNF-alpha. GMH enhanced protein level of TNF-alpha while CD200Fc attenuated this induction. Data are expressed as mean ± SD, *P < 0.05 vs Sham, ^#P < 0.05 vs GMH + Vehicle, ^&P < 0.05 vs Treatment, n = 6/group, one-way ANOVA followed by the Tukey test.

Discussion

GMH is a common consequence of premature newborns and there is still no effective treatment in the clinical settings other than surgical shunting to ameliorate hydropcephalus.³⁵ GMH elicits a robust acute inflammatory cascade, contributing to long-term morphological and functional impairment.⁴ Therefore, targeting short-term neuroinflammation can be a critical strategy for the treatment of GMH.³⁶ Previously, we developed and characterized a novel GMH model using collagenase injection in neonatal rats, which mimics the motor deficits and ventricular dilation in human preterm newborns. CD200 and its receptor CD200R are appreciated as critical endogenous inflammatory regulatory systems.^21,37 Thus, in this study, we evaluated the protective effects of CD200Fc in this GMH model with a particular interest in neuroinflammation. We first examined the expression profile of endogenous CD200 and CD200R1 following GMH and then assessed the therapeutic effects of exogenous CD200Fc using both short-term and long-term neurobehavioral tests. In addition, CD200R1 siRNA and Dok1 siRNA were employed to establish the signaling pathway of CD200R1. We have observed that (1) the endogenous expression levels of CD200 and its receptor CD200R1 decreased after GMH and that CD200R1 was expressed on microglia cells. (2) Administration of external CD200Fc preserved BBB integrity and improved short-term neurological functions, accompanied by reduced IL-1beta and enhanced ZO-1 expression. (3) CD200Fc leads to long-term functional improvement on Day 28 after GMH. (4) CD200R1 and Dok1 siRNA abrogated the beneficial effects of CD200Fc, indicating that CD200R1 and Dok1 are downstream mediators of CD200FC. These data indicate that exogenous CD200 plays a beneficial role after GMH by mitigating neuroinflammation and that these effects are possibly mediated by CD200R1/Dok1 pathway.

In the present study, our results are consistent with several previous observations that CD200Fc plays an anti-inflammatory role in the brain. Cox et al.¹⁹ showed that CD200 decreased microglial activation in the hippocampus of aged rats. On the contrary, using a CD200 neutralizing antibody in an experimental allergic encephalomyelitis rat model resulted in significantly greater disease scores and enhanced inflammation and aggregates of activated microglia were seen in the spinal cord in CD200 gene-deficient mice.^12,38

In the central nervous system, CD200 is localized on neurons, endothelial cells and subsets of astrocytes while CD200R1 is mainly expressed on microglia and to a lesser extent, on astrocytes and neurons.³⁹ A report from Dentesano showed that CD200 expression at 24 h after LPS/IFN-c challenge was not modified in neuron-enriched cultures.⁴⁰ However, CD200 expression was increased in primary mixed glial cultures in response to LPS/IFN-c. It indicates that neurons and glia cells respond differently in terms of CD200 expression. Our time course results showed that CD200 and CD200R1 levels were both decreased at 24 h after GMH. A possible speculation for the decrease of CD200 is that neurons go under much worse injury in experimental GMH, of which the damaging factors also include hypoxia, acidosis besides inflammation. Since CD200 is primarily produced in neurons, neuronal CD200 expression might decrease with the greater neuronal damage. The decrease in overall CD200 level might be the consequence of glial CD200 not being able to compensate for the loss of neuronal CD200 level.

The binding of neuronal CD200 to microglial CD200R1 through N-terminal amino acid sequences results in the activation of microglial CD200R through cell–cell contact. CD200–CD200R1 interaction involves the activation of the adaptor proteins Dok1, Dok2, and RasGAP, leading to the inhibition of Ras activation with multiple downstream effects to trigger anti-inflammatory signals (Supplementary Figure 1).^21,37 To establish a potential mechanism for the actions of CD200–CD200R, similar to other studies using siRNA to explore the molecular pathways,^41,42 we knocked down CD200R1 and Dok1 by CD200R1 siRNA and Dok1 siRNA, respectively. We found that the beneficial effect of CD200Fc was reversed. Specifically, after CD200R1 and Dok1 were inhibited by siRNA, the inflammatory response was restored. This observation indicated that CD200R1 and Dok1 are involved in the signaling pathways of CD200–CD200R1.

In our study, we found BBB permeability decreased at 24h with the high-dose CD200Fc treatment through the Evans Blue extravasation assay, along with increased ZO-1 protein expression at the same time point. These results showed CD200Fc preserved BBB-related proteins and protect BBB function. Neuroinflammation caused by microglial activation is associated with increased BBB permeability.^43,44 Previous reports also showed that classical activation of microglia in CD200-deficient mice increased BBB permeability and infiltration of peripheral cells.²⁴ Although we only focused on microglia in this study, we do not exclude the possibility that CD200Fc activates other pathways in other cell types to preserve BBB integrity, which will be explored in our future studies.

There are no reports that CD200Fc can cross BBB. In addition, the molecular weight of CD200Fc in this study is 37 kDa. Therefore, it cannot cross the BBB. Our peers have tried intrahippocampal application and intrathecal application. They found that CD200 fusion protein decreases microglial activation.^19,29 In this study, we used the i.c.v. injection approach, which is easy to perform and control, to first establish the protective effects of CD200–CD200R in GMH and its underlying mechanisms. Although i.c.v. injection is invasive, premature infants with GMH frequently get intraventricular shunts/reservoirs.⁴⁵ Thus, i.c.v. administration may certainly be possible and clinically relevant. In addition, intrathecal or intranasal applications can also be used in clinics with pharmaceutical modifications to help CD200Fc cross the BBB.

One of the limitations of our current study is that we only focused on CD200R1 and Dok1. However, our results do not exclude the potential involvement of CD200Rs and Dok2 or RasGAP in the inflammation after GMH,^21,38 which may be further evaluated in our future research.

Conclusion

We demonstrated for the first time that the CD200/CD200R1/Dok1 signaling pathway attenuates neuroinflammation after GMH. This pathway provides a potential molecular target to reduce inflammation in GMH or other types of brain injuries.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: NIH NINDS NS078755 to JHZ.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors' contributions

Z.F. and L.Y. conceived, designed the experiments, carried out behavioral tests, western blot, and immunohistochemistry, and wrote the manuscript. D.K. and J.F. carried out intracerebroventricular drug administration, behavioral tests, western blot; Y.D. and C.Y. carried out Evans Blue extravasation assay. J.T. and J.D. conceived and participated in acquiring and analyzing the presented data. J.Z. conceived, designed, and coordinated the study, and helped to draft the manuscript. Z.F., Y.D. and D.K. worked on manuscript revision. All authors read and approved the final manuscript.

Supplementary material

Supplementary material for this paper can be found at the journal website: http://journals.sagepub.com/home/jcb