NK cells inltrating in the central nervous system aggravate brain injury of mice caused by Angiostrongylus cantonensis infection

Background Angiostrongylus cantonensis (A. cantonensis), is a food-borne zoonotic parasite that can cause central nervous system (CNS) injury characterized by eosinophilic meningitis. However, the pathogenesis of the neurological impairments caused by A. cantonensis infection has not been well elucidated. Natural killer cells (NK cells) are unique innate lymphocytes important in early defense against pathogens. It was reported that NK cells could migrate to the CNS after brain injury. The aim of the present study was to investigate the role of NK cells in brain injury caused by A. cantonensis infection. Methods Mouse model of A. cantonensis infection was established by intragastric administration of third-stage larvae. Neurological impairments were evaluated by Longa’s score, Clark’s general score and Clark’s focal score. Histopathological changes were observed by hematoxylin and eosin staining. The expression of cytokines at gene and protein levels was analyzed by PCR and ELISA, respectively. Inltration of NK cells in the CNS was detected by immunohistochemistry and ow cytometry. Depletion of NK cells in infected mice was caused by tail vein injection of anti-asialo GM1 rabbit serum, and adoptive transfer of NK cells was performed by tail vein injection of puried splenic NK cells. NK cell-mediated cytotoxicity against YAC-1 cells was detected by LDH release assay. The cytokine production ability was determined by intracellular ow cytometry and ELISA. Results Mice developed brain inammation and neurological impairment after A. cantonensis infection. The inltration of NK cells in the CNS of A. cantonensis-infected mice was observed on 14 dpi and reached the peak on 22 dpi. Compared with the normal splenic NK cells, the CNS-inltrated NK cells of infected mice expressed lower levels of CD69, NKp46 and NKG2D, but higher levels of NKG2A, and showed enhanced cytotoxicity and increased IFN-γ and TNF-α production ability. Depletion of NK cells alleviated brain injury, whereas adoptive transfer of NK cells exacerbated brain damage in A. Conclusions Our results demonstrate that NK cells inltrate into the CNS and aggravate the brain damage after A. cantonensis infection. The ndings improve the understanding the pathogenesis of angiostrongyliasis and expand the therapeutic intervention in CNS disease. Survival curve comparison was determined using Log-rank Test. Comparison of the neurological impairment scores was performed using non-parametric test. The difference between two groups was compared using independent-samples T test. Multiple comparison procedures were carried out with one-way analysis of variance (ANOVA). The correlation between the percentage and number of NK cells in different tissues was analyzed by linear correlation. The data are presented as the mean ± standard deviation (SD). A P value < 0.05 was considered statistically signicant. A.cantonensis: Angiostrongylus cantonensis; BBB: Brain-blood Barrier; bNK: NK cells in brain; bmNK: NK cells in bone marrow; CNS: Central nervous system; DAB: diaminobenzidine; EAE: Experimental autoimmune encephalomyelitis; H&E: Hematoxylin and eosin staining; ICH: intracerebral hemorrhage; IHC: Immunohistochemistry; L3: Third-stage larvae; LAMP-1: Lysosome associated membrane protein-1; MACS: Magnetic cell sorting system; NK cells: Natural killer cells; NKP: NK precursor cells; pbNK: NK cells in peripheral blood; PBMCs: Peripheral blood mononuclear cells; pMCAO: permanent middle cerebral artery occlusion; SFV; Semliki Forest virus; sNK; NK cells in spleen. not have signicant changes. Representative histopathological sections of brain tissue with H&E and IHC staining (j) showed alleviated inammation and fewer inltrating NK cells in the brain of NK-depleted mice. Images are shown at 100 ×, 200 × and 400 × magnication (Scale bar, 50-100 µm). The gene (k-m) and protein levels (n-p) of inammatory cytokines IL-1β, IL-6 and TNF-α in brain tissue reduced after NK cell depletion measured by qRT-PCR and ELISA. Data are expressed as the means ± SD. Data showed represent analysis from two independent experiments with three to twelve mice per group. Survival curve comparison was determined by Log-rank Test. Comparison of the neurological impairment scores was compared by non-parametric test. Multiple comparisons of the percentage and number of NK cells, body weight and expression of cytokines were performed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Uninfected, normal mice; Infected, mice infected with A. cantonensis; NK Depleted, infected mice depleted NK cells by injection of anti-asialo GM1 rabbit serum; Depleted Control, infected mice given normal rabbit serum as control.


Background
Angiostrongylus cantonensis (A. cantonensis), is a food-borne zoonotic parasite that can cause damage to the central nervous system (CNS) [1]. It was rst discovered in the pulmonary arteries and hearts of domestic rats in Guangzhou, China, by Chen in 1935 [2]. So far, nearly 3000 cases of human angiostrongyliasis have been documented worldwide [3]. With the development of global logistics transportation and climate warming, A. cantonensis has spread from its traditional endemic regions of Southeast Asia and the Paci c islands to the American continent, Europe, Africa and Australia [4][5][6][7][8][9].
Therefore, angiostrongyliasis has become a potentially fatal globally emerging infectious disease.
Humans and mouse are both non-permissive hosts of A. cantonensis and become infected via ingestion of raw or undercooked intermediate hosts including snails or slugs, or vegetables or water contaminated by the infective third-stage larvae [3]. After penetrating the intestinal wall, the larvae migrate in the body with the ow of blood, and nally settle in the CNS. These larvae in non-permissive hosts cannot develop into adults, but can survive in the form of larvae for a long time [10]. Most patients when infected with A. cantonensis develop eosinophilic meningitis and common clinical symptoms include headache, fever, neck stiffness, paresthesia and vomiting [11]. However, the pathogenesis of angiostrongyliasis is not fully understood. Mechanical damage to the CNS caused by the larvae' movements, in ammation and immune response caused by the larval secretion and excreta, may be involved [11][12][13].
Natural killer cells (NK cells) are a type of cytotoxic lymphocytes critical to the innate immune system [14]. NK cells lack T and B cell receptors and undergo activation without antigen presentation. They are able to rapidly kill target cells by cytotoxicity and produce an abundance of cytokines [15]. NK cells are dispersed throughout lymphoid and non-lymphoid tissues. It was reported that NK cells could migrate to the CNS under several pathological conditions, such as brain ischemia, traumatic injury, or infections [16]. However, very little is known about whether NK cells are involved in the CNS damage caused by A. cantonensis infection.
The objective of this study was to investigate the role of NK cells in A. cantonensis infection. Our ndings will be helpful to further elucidate the pathogenesis of angiostrongyliasis and expand therapeutic intervention in CNS disease.

Animal experiments
A total of 300 female BALB/c mice (6-8 weeks old and weighing 18-20 g) were purchased from the Animal Core Facility of Nanjing Medical University (China), maintained in a speci c pathogen-free environment, and provided unlimited access to food and water. All experiments were performed in strict compliance with the institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval No. IACUC-1812040). Each mouse was infected with 20 A. cantonensis third-stage larvae (L3) by intragastric administration. L3 were isolated from A. cantonensis-infected Biomphalaria glabrata using the method described previously [13]. The mice were euthanized on 10, 14, 18, 22, and 26 days post-infection (dpi).

Experimental Grouping
The experiment was grouped according to different objectives.
To detect the effect of A. cantonensis infection on mice and the percentage and number of NK cells, the mice were divided into ve groups according to the time of infection with three to twelve mice per group: 0, 10, 14, 18 and 22 dpi. When detecting the phenotypic and functional changes of NK cells, cells were isolated from mice on 18 dpi and divided into three groups with three to four mice per group: splenic NK cells of uninfected mice (uninfected sNK), splenic NK cells of infected mice (infected sNK) and brain NK cells of infected mice (infected bNK).
In the experiment of NK cell depletion or adoptive transfer, the mice were euthanatized on 18 dpi and divided into four groups with three to twelve mice per group: uninfected group, infected group, NK depleted/transferred group and depleted/transferred control group.

Neurological Impairment Evaluation
Longa's score, Clark's general score and Clark's focal score were used to evaluate the neurological impairment of mice infected with A. cantonensis. Longa's score is based on a ve-point scale, where 0 point indicates no neurologic de cit, 1 point (failure to extend forepaw fully) a mild focal neurologic de cit, 2 point (circling to one side) a moderate focal neurologic de cit, 3 point (falling to one side) a severe focal de cit, and animals with 4 point cannot walk spontaneously or lose consciousness [17]. Clark's general score includes hair, ears, eyes, posture, autonomous movement, and epileptic seizures.
Clark's focal score comprises of body symmetry, gait, climbing, rotation test, forelimb symmetry and beard reactivity [18]. Clark's general score and Clark's focal score are between 0 and 28. The higher the score, the more serious the neurological impairment is. The mice were scored by two technicians using a blind method.

Histopathological Examination
Mice were perfused transcardially with 0.9% sodium chloride followed by 4% paraformaldehyde after anesthetized with 2% pentobarbital sodium (Sigma-Aldrich, USA). Brain samples were collected, xed in 10% neutral formalin, embedded in para n, and cut into 3 µm-thick sections. Brain sections were then depara nized in xylene, rehydrated via graded alcohols and stained with hematoxylin and eosin (H&E) (Biosharp, Wuhan, China). The sections were observed and photographed under a light microscope (Leica, Heidelberg, Germany).
For immunohistochemistry (IHC) analysis, brain sections were subjected to antigen retrieval by boiling the slices in citrate buffer (pH 6.0) with high heat for 15 min. Then sections were treated with 3% H 2 O 2 for 10 min to remove endogenous peroxidase, blocked with 5% rabbit serum at room temperature for 20 min, and incubated with rabbit anti-mouse CD49b monoclonal antibody (mAb) (Abcam, Cambridge, UK) at 4 °C overnight. After being washed in PBS, the sections were incubated with an HRP-conjugated secondary antibody (DAKO, Glostrup, Denmark) at room temperature for 15 min and then stained with 3, 3'-diaminobenzidine (DAB) for 10 min. Haematoxylin was used for cell nuclei detection. The sections were visualized and digitally scanned with a light microscope.
Quantitative Reverse Transcription PCR (QRT-PCR) Total RNA was extracted from mouse brains using TRIzol Reagent (Thermo Fisher Scienti c, USA) and reverse-transcribed to cDNA using a PimerScript™ RT Master Mix (TaKaRa, Kusatsu, Japan). qRT-PCR was performed on the LightCycler480® Real-Time PCR System (Roche, Reinach, Switzerland) with the RealUniversal Color PreMix (SYBR Green) (Tiangen, Beijing, China), in accordance with the manufacturer's instructions. The primer sequences were shown in Table 1. The mRNA levels of these genes were measured by the Ct value (threshold cycle), and the relative expression levels were calculated with the 2 −ΔΔCt method.

Cell Isolation
Blood was collected by eyeball bleeding and gathered in tubes with 1% heparin sodium (Sigma-Aldrich, USA). The mice were anesthetized and perfused as described previously. And then, the brain, spleen, tibia, and femur were collected from the mice respectively.
Brain tissues were homogenized in grinders and ltered through a 70-µm cell strainer. And then cell pellets were resuspended in 30% Percoll (GE Healthcare, Pittsburgh, USA) and centrifuged against 70% Percoll. The cells between the 30-70% Percoll interfaces were collected as the brain mononuclear cells. Spleen was grinded with a syringe core and ltered through a nylon membrane. Erythrocytes were lysed and removed using Red Cell Lysis Buffer (Beyotime, Shanghai, China). Blood was double diluted and layered on the Ficoll-Paque (GE Healthcare, Pittsburgh, USA). After centrifugation, peripheral blood mononuclear cells (PBMCs) were distributed between plasma and Ficoll-Paque. PBMCs were transferred and resuspended in PBS. The tibia and femur bones were used to prepare bone marrow cells. The medullary cavity was washed repeatedly with RPMI-1640 (Gibco B, Gaithersburg, MD, USA) by syringe and bone marrow cells were collected and separated from erythrocytes.
NK cells were puri ed from brain mononuclear cells and splenic lymphocytes using a magnetic cell sorting system (MACS) incorporating anti-mice CD49 MicroBeads (MiltenyiBiotec, Bergisch Gladbach, Germany), following the manufacturer's instructions. The purity of CD3 − CD49 + NK cells after sorting was over 90% detected by FCM (Additional le 1: Figure S1).

Flow Cytometry (FCM)
Cells isolated from the brain, spleen, peripheral blood, and bone marrow were prepared to single cell The absorbance of the samples was measured at 490 nm by an ELISA reader. The percentage of NK cellmediated cytotoxicity was calculated by ODs using the following equation: Cytotoxicity (%) = (effector/target cell mix -effector cell control -low control) / (high control -low control) × 100.

Detection Of NK Cells Cytokine Secretion
Freshly puri ed NK cells were cultured in 24-well plates at a density of 1 × 10 6 /mL in the presence of 1 ng/mL IL-12 (PeproTech, Rocky Hill, NJ, USA). After 24 h of incubation, the culture supernatants were harvested. The concentrations of TNF-α and IFN-γ in supernatants were detected using Mouse TNF-α or IFN-γ High Sensitivity ELISA Kits (MultiSciences, Hangzhou, China) according to the instructions of the manufacturer.
Depletion Of NK Cells 5-7 Ganglio-N-tetraosylceramide (asialo GM1) is a glycolipid expressed on NK cells in mice, rats, and humans. Depletion of NK cells in vivo in infected mice was induced by tail vein injection of anti-asialo GM1 rabbit serum (Wako Pure Chemical Industries, Japan) according to the instructions from manufacturer and other researchers [19,20]. Injection time and dose were shown in Table 2. Normal rabbit serum (Abbkine, California, USA) were given to the infected mice at the same time as control. To evaluate the effect of NK cell depletion, the mice were euthanatized at 18 dpi and the percentage and number of NK cells in brain and spleen were analyzed by FCM.  Adoptive Transfer Of NK Cells The splenic NK cells from normal mice were puri ed using MACS and were suspended in PBS. The NK cells (1 × 10 6 /mouse in 200 µL PBS) were transferred to the infected mice by tail vein injection on 12 dpi according to the protocols published by other investigators [21]. The respective diluents were injected to the control mice simultaneously. To evaluate the effect of adoptive transfer, the mice were euthanatized at 18 dpi and the percentage and number of NK cells in brain and spleen were analyzed by FCM.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, USA). Survival curve comparison was determined using Log-rank Test. Comparison of the neurological impairment scores was performed using non-parametric test. The difference between two groups was compared using independent-samples T test. Multiple comparison procedures were carried out with oneway analysis of variance (ANOVA). The correlation between the percentage and number of NK cells in different tissues was analyzed by linear correlation. The data are presented as the mean ± standard deviation (SD). A P value < 0.05 was considered statistically signi cant.

A. cantonensis infection causes neurological impairment and brain in ammation in mice
We constructed a mouse model of A. cantonensis infection by intragastric administration of third-stage larvae. To evaluate the pathological damage caused by A. cantonensis infection, the survival rate, body weight, neurological function, histopathology, and expression of cytokines in brain tissue were detected on 0, 10, 14, 18 and 22 dpi.
The survival rate of mice decreased gradually with the extension of infection time (Fig. 1a). Only 50% of the mice survived on 22 dpi and the survival rate of mice was merely 8.33% on 30 dpi. The weight of infected mice decreased signi cantly compared with that of uninfected mice on 18 dpi and 22 dpi (Fig. 1b).
After infection, various symptoms of neurologic de cit appeared in mice including erect hair, arched back, falling to one side when walking, and circling to one side continuously when their tails were lifted. Some mice even could not walk spontaneously, became blind or lost consciousness (Fig. 1c). The Longa's score, Clark's general score and Clark's focal score used to evaluate neurological impairment elevated signi cantly on 18 and 22 dpi compared with that of 0 dpi (Fig. 1d-f).
Then we observed the histopathological changes of brain tissue in mice. On 10 dpi, there were a few red bleeding spots in the brain tissue, and 1-2 brown, slim, and curving fourth-stage larvae(L4)appeared on the surface. The increased hemorrhage and more invasive larvae were found in the brain tissue from 14 to 22 dpi (Fig. 1g). And then, we prepared the brain tissue sections with H&E staining. On 14 dpi, the meninges became thicker and a few in ammatory cells in ltrated under the meninges. On 18 dpi, the number of in ltrating in ammatory cells increased. On 22 dpi, the meninges were damaged, a large number of in ammatory cells in ltrated under the meninges, and multiple areas of red bleeding area were found (Fig. 1h).
We further examined the gene and protein levels of various in ammatory cytokines in brain tissue. The mRNA levels of IL-1β, IL-6, TNF-α and the protein levels of IL-6 increased signi cantly on 14, 18 and 22 dpi, and the protein levels of IL-1β and TNF-α elevated signi cantly on 14 and 18 dpi, compared with that on 0 dpi (Fig. 1i-n).
NK cells in ltrate into the CNS of mice infected with A. cantonensis H&E staining showed that many in ammatory cells in ltrated into the CNS after A. cantonensis infection.
In order to detect the presence of NK cells in these in ammatory cells, we performed IHC staining on brain tissue sections using anti-CD49b mAb as NK cell marker. Figure 2a, b showed that no NK cell was observed in the CNS on 0 dpi and 10 dpi. On 14 dpi, a small number of NK cells stained brown appeared under the meninges indicating that NK cells began to in ltrate into the CNS. On 18 dpi, more NK cells appeared in the CNS and the number of CNS-in ltrated NK cells peaked on 22 dpi.
To monitor NK cells quantitatively, brain mononuclear cells were isolated and analyzed by FCM at various time points. As shown in Fig. 2c-e, almost no NK cells were detected in the brain tissue at 0 dpi and 10 dpi. NK cells appeared in the CNS from 14 dpi and the percentage and number of CNS-in ltrated NK cells increased gradually with the extension of infection time. The highest percentage of CNS-in ltrated NK cells appeared on 22 dpi (22 dpi vs 0 dpi: 17.47 ± 6.11% vs 0.45 ± 0.12%, P < 0.001) and the maximum number of CNS-in ltrated NK cells were found on 18 dpi (18 dpi vs 0 dpi: 1.92 ± 0.43 × 10 5 vs 696.9 ± 617.2, P < 0.001).
To elucidate the origin of NK cells in the CNS, we further analyzed the percentage and number of NK cells in the splenic lymphocytes, peripheral blood mononuclear cells and bone marrow cells by FCM. The results showed that the percentage and number of NK cells in splenic lymphocytes decreased on 14, 18 and 22 dpi. (Fig. 3a, d, e). So did the number of NK cells in PBMCs from 10 dpi to 22 dpi (Fig. 3b, e, f). Furthermore, the percentage and number of sNK (NK cells in spleen) were negatively correlated with those of bNK (NK cells in brain) (r =0.79, P < 0.01; r =0.85,P < 0.01) (Fig. 3g, h). The percentage and number of pbNK (NK cells in PBMCs) and bNK were also negatively correlated, but there was no statistical signi cance (Fig. 3i, j).

CNS-in ltrated NK cells of A. cantonensis-infected mice have elevated cytotoxicity and secretory ability
We further detected the phenotypic and functional changes of NK cells after A. cantonensis infection. We measured the expression of activation marker CD69, activated receptor NKp46 and NKG2D, and inhibitory receptor NKG2A on NK cells by FCM. Figure 5a-g revealed that infected bNK expressed lower levels of CD69, NKp46 and NKG2D, but higher levels of NKG2A, and infected sNK expressed lower levels of CD69, NKp46, but higher levels of NKG2A, compared with uninfected sNK. Although the phenotypic changes of infected bNK were greater than those of infected sNK, there was no statistical difference between them.
Then we detected NK cell-mediated cytotoxicity against YAC-1 cells by LDH release assay. As shown in Fig. 6a, infected bNK and infected sNK had enhanced cytotoxicity, compared with uninfected sNK (Effect cells: Target cells = 20:1, 19.37% vs 10.59% vs 8.35%). Furthermore, the expression of CD107a, a surface marker of NK cell degranulation, on infected bNK and infected sNK was up-regulated compared with that of uninfected sNK (Fig. 6b, c).
The ability of NK cells to secrete cytokines was determined by two methods: ELISA (secretory levels) and FCM (intracellular levels). Firstly, we detected the concentrations of TNF-α and IFN-γ in the culture supernatant of NK cells after IL-12 stimulation using ELISA. The levels of TNF-α and IFN-γ secreted by infected bNK and infected sNK were signi cantly higher than those of uninfected sNK (Fig. 6d).
Subsequently, puri ed NK cells were stimulated and measured by FCM. The percentage of IFN-γ + cells in infected bNK was higher than that of uninfected sNK, while the percentage of TNF-α + or IFN-γ + cells in infected sNK did not change signi cantly (Fig. 6e, f).

NK cells aggravate brain injury of mice caused by A. cantonensis infection
We designed NK cell depletion and adoptive transfer experiments to elucidate the role of NK cells in brain injury caused by A. cantonensis infection.
Firstly, NK cells were depleted by tail vein injection of anti-asialo GM1 serum to infected mice. The percentage and number of NK cells in brain and spleen of infected mice signi cantly decreased after NK cell depletion (Fig. 7a-d). The survival rate of NK-depleted mice increased (P < 0.01) (Fig. 7e), body weight elevated (17.35 ± 1.38 g vs 15.61 ± 1.1 g, P < 0.05) (Fig. 7f), neurological impairment score decreased slightly (Fig. 7g-i), brain in ammation alleviated (Fig. 7j), and the levels of in ammatory cytokines in brain tissue reduced(IL-1β 25 (Fig. 7k-p), compared with the infected mice on 18dpi. These results showed that depletion of NK cells alleviated brain injury in A. cantonensis-infected mice.
And then puri ed splenic NK cells were transferred to infected mice by tail vein injection. The percentage of NK cells in brain and spleen of infected mice increased signi cantly after adoptive transferring NK cells (Fig. 8a-d). Compared with the infected mice, the survival rate, body weight and neurological impairment score of NK-transferred mice did not change signi cantly (Fig. 8e-i), but brain in ammation aggravated (Fig. 8j), and the concentrations of in ammatory cytokines in brain tissue elevated (IL-1β 36.36 ± 1.89 pg/mg vs 30.75 ± 2.83 pg/mg, P < 0.05; IL-6 18.08 ± 0.55 pg/mg vs 15.08 ± 1.13 pg/mg, P < 0.01; TNF-α 76.84 ± 1.21 pg/mg vs 64.68 ± 4.34 pg/mg, P < 0.01) (Fig. 8k-p) on 18dpi. It was suggested that adoptive transfer of NK cells exacerbated brain damage in mice caused by A. cantonensis.

Discussion
Angiostrongylosis, a food-borne parasitic disease, is caused by the larvae of A. cantonensis in the host's central nervous system [22]. NK cells are important innate immune effector cells. They can be swiftly mobilized by danger signals and are among the earliest arrivals at target organs against pathogen infection [23]. However, the role of NK cells in the CNS damage caused by A. cantonensis infection remains elusive. Our previous work has reported that NK cells in the spleen and peripheral blood showed quantitative reduction and functional changes in an A. cantonensis-infected mice model [24]. In the current study, we focused on the NK cells in the CNS. We found a large number of NK cells in ltrated into the CNS of mice after A. cantonensis infection and these CNS-in ltrated NK cells had elevated cytotoxicity and secretory ability. Moreover, we demonstrated that NK cells aggravated brain injury of mice caused by A. cantonensis infection.
Mice and humans are both non-permissive hosts of A. cantonensis, and the pathogenic process is comparatively similar [25]. Consistent with other reports [26][27][28], we successfully constructed a mouse model infected with A. cantonensis and observed serious neurological damage from 18 dpi to 22 dpi. What is noteworthy in this study is that we con rmed that NK cells in ltrated into the CNS after A. cantonensis infection. The results of IHC and FCM showed that NK cells began to appear in the brain tissues at 14 dpi. The percentage and number of NK cells increased gradually with the extension of infection time until 22 dpi. The CNS, including the brain and spinal cord, is considered as an immune privileged organ because of the low permeability of the brain-blood barrier (BBB). However, it is conceivable that peripherally activated lymphocytes, including NK cells, might also be able to penetrate the BBB and in ltrate into the CNS under some pathological conditions. In human ischemic brain tissue and a permanent middle cerebral artery occlusion (pMCAO) mouse model, in ltration of NK cells into the ischemic infarct region are observed [29][30][31]. NK cells are activated in the periphery and then migrated into the CNS of experimental autoimmune encephalomyelitis (EAE) mice [32]. NK cells can be detected in mouse CNS tissues during a variety of infections, including Semliki Forest virus (SFV) [33], murine coronavirus [34] and L. monocytogenes [35]. NK cells are also recruited to the CNS in glioma-bearing mice and constitute approximately 50% of all leukocytes in the CNS [36].
To explore the origin of NK cells in the CNS, we examined the distribution of NK cells in A. cantonensisinfected mice. The results showed that the percentage and number of NK cells in spleen and in peripheral blood both decreased, consistent with our previous study [24], while the percentage of NK cells in bone marrow increased after A. cantonensis infection. In addition, the percentage and number of splenic NK cells were negatively correlated with those of brain NK cells, while the percentage of NK cells in bone marrow was positively correlated with brain NK cells. It was suggested that the CNS-in ltrated NK cells probably were related with the increased hematopoiesis of bone marrow and migration of peripheral NK cells after A. cantonensis infection. NK cells originate from bone marrow, are mainly distributed in peripheral blood and spleen, and some lymphatic tissues [15]. However, the distribution of NK cells is not static because these cells can recirculate between organs. NK cells can respond to a large array of chemokines and be recruited to distinct sites in several pathological circumstances [16]. The detailed tra cking patterns of NK cells are not very well characterized. Nevertheless, it appears that chemokines produced by cells that are unique to speci c organs may have a role in orchestrating NK cell migration to each organ [16]. It was reported that NK cells might be recruited to the CNS by chemokines such as CX 3 CL1 produced by neurons [37] and CCL2 and CXCL10 produced by microglia, astrocytes or in ltrating in ammatory cells [29,38]. The mechanism of recruitment of NK cells to the CNS after A. cantonensis infection needs to be further studied.
NK cells are a kind of special lymphocytes, which can lyse the target cells directly and produce various cytokines to regulate immune response [39]. To further identify the characteristics of the CNS-in ltrated NK cells, we detected the phenotype and function of NK cells after A. cantonensis infection. The results showed that the expression of activation molecule CD69, activating receptor NKp46 and NKG2D on the CNS-in ltrated NK cells of infected mice was decreased compared with the splenic NK cells of uninfected mice, while the expression of inhibitory receptor NKG2A increased. Consistent phenotypic changes were observed in the splenic NK cells of infected mice except for NKG2D. CD69 is one of the early activation markers on NK cells and is involved in cell proliferation and signal transduction. Meanwhile, CD69 a novel regulator of the immune response, which can inhibit the cytotoxicity of NK cells by inducing the production of TGF-β. It was demonstrated that the administration of anti-CD69 mAbs can activate resting NK cells, resulting in a substantial increase in both NK-cell cytolytic activity and IFN-γ production [40]. perforin/granzyme, Fas/FasL, TNF-α/TNFR-I and other pathways [15]. In our study, we found that the CNS-in ltrated NK cells of infected mice had an enhanced cytotoxicity against YAC-1 cells with higher expression of CD107a. CD107a, also known as Lysosome associated membrane protein-1 (LAMP-1), is a marker for degranulation of NK cells and CD8 + T cells [41]. It is reported that CD107a expression correlates with NK cell-mediated lysis of target cells and is a sensitive marker of NK cell activity [42]. NK cells can also produce a variety of cytokines in response to activation signaling to mediate immunomodulatory effects. Our results showed that the CNS-in ltrated NK cells of infected mice produced higher levels of TNF-α and IFN-γ than splenic NK cells of uninfected mice. TNF-α is a proin ammatory cytokine that is not only cytotoxic to tumor cells but also involved in mediating antipathogen immune responses. IFN-γ is an important immunomodulator with the function of activating macrophages, promoting MHC molecular expression and antigen presentation, and plays an important role in defending viral, bacterial and parasitic infections [43,44]. Taken together, the CNS-in ltrated NK cells in A. cantonensis-infected mice showed stronger activity with enhanced cytotoxicity and elevated production of TNF-α and IFN-γ. There are no NK cells in the steady-state CNS, but NK cells might migrate into the CNS under certain pathological conditions such as A. cantonensis infection. After homing to the in amed CNS, NK cells become receptive to an array of cellular components that they have not encountered in the periphery. These include astrocytes, microglia, neurons, and eosinophils, which release numerous soluble factors with diversi ed and perhaps coordinated effects on NK cells [16]. The exact cellular and molecular interactions that shape the phenotype and function of NK cells in the CNS still need to be determined.
We then investigated the role of NK cells in the brain damage caused by A. cantonensis infection using NK cell depletion and adoptive transfer experiments. The changes in NK cell percentage and number con rmed that NK cell depletion and adoptive transfer were effective. Our results showed that the survival rate and body weight increased, nerve injury and brain in ammation decreased after NK depletion in A. cantonensis-infected mice. On the contrary, the in ammation in brain aggravated after adoptive transfer of NK cells. We speculated that the CNS-in ltrated NK cells might play a role in exacerbating brain injury after A. cantonensis infection. Many studies have reported that NK cells can rapidly accumulated into the CNS under pathological conditions. However, the role of NK cells in brain injury diseases remains complex and paradoxical. Zhiguo Li [31] identi ed NK cells in ltrates into the CNS during early stages of intracerebral hemorrhage (ICH). These in ltrating NK cells exacerbate BBB disruption and brain edema via cytotoxicity toward cerebral endothelial cells and recruitment of neutrophils. Gan  injury, which may be related to the initial factors of the primary disease, the time of immune response, and the overall in ammatory process [30]. After brain injury, brain-intrinsic cells, including microglia, astrocytes and neurons, secrete various chemokines which recruiting a series of in ammatory cells. As an important type of immune cell, peripheral resident NK cells are activated and migrate to brain tissue [16]. NK cells in ltrating into the CNS can build a bridge between immune system and nervous system, which not only produce cytotoxicity or cytolysis to pathogens, but also interact with other cells to affect the progress of brain injury. In the process of A. cantonensis infection, NK cells in ltrating in brain tissue have stronger killing function and secrete more IFN-γ and TNF-α. On the one hand, these CNS-in ltrated NK cells were bene cial to clearance of pathogens, on the other hand, they were also involved in the death of neural cells and aggravated brain damage.
Currently, the treatment for angiostrongyliasis includes supportive treatment and corticosteroid therapy [46]. The use of anthelmintic drugs, such as albendazole and mebendazole, to kill worms remains controversial. Because the dead worm lysis in the CNS might cause severe in ammatory response and further damage [10]. Meanwhile, Patients under the treatment of high-dose corticosteroids would experience immune suppression [47]. Therefore, it is urgent to develop new therapeutic interventions for angiostrongyliasis. We hypothesized that selective reduction of NK cell in ltration into the CNS or control their activation may help to alleviate the brain injury caused by A. cantonensis infection, which need to be further investigated in our future work.

Conclusion
In summary, our study demonstrates that NK cells in ltrate into the CNS of A. cantonensis-infected mice. These CNS-in ltrated NK cells display enhanced cytotoxicity and secretary ability, which may contribute to aggravate brain damage. Our ndings not only enrich the understanding of A. cantonensis pathogenesis but also provide a clue to novel potential therapeutic strategies against CNS disease. After NK cell depletion, the survival rate of infected mice (e) increased, body weight (f) elevated, while neurological impairment scores (g-i) did not have signi cant changes. Representative histopathological sections of brain tissue with H&E and IHC staining (j) showed alleviated in ammation and fewer in ltrating NK cells in the brain of NK-depleted mice. Images are shown at 100 ×, 200 × and 400 × magni cation (Scale bar, 50-100 µm). The gene (k-m) and protein levels (n-p) of in ammatory cytokines IL-1β, IL-6 and TNF-α in brain tissue reduced after NK cell depletion measured by qRT-PCR and ELISA. Data are expressed as the means ± SD. Data showed represent analysis from two independent experiments with three to twelve mice per group. Survival curve comparison was determined by Log-rank Test. Comparison of the neurological impairment scores was compared by non-parametric test. Multiple comparisons of the percentage and number of NK cells, body weight and expression of cytokines were performed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Uninfected, normal mice; Infected, mice infected with A. cantonensis; NK Depleted, infected mice depleted NK cells by injection of anti-asialo GM1 rabbit serum; Depleted Control, infected mice given normal rabbit serum as control.