Anti-IL-23 exerted protective effects on cerebral ischemia-reperfusion injury through JAK2/STAT3 signaling pathway

Ischemia-reperfusion is frequently occurred in ischemic cerebral vascular disease, during which process the inammatory signaling played essential roles. The aim of study was to discover the ecacy of the antibody to a key immune cytokine IL-23 (anti-IL-23) for the therapy of cerebral ischemia-reperfusion injury. Methods We established the cerebral ischemia-reperfusion injury model by middle cerebral artery occlusion (MCAO). Anti-IL-23 injection attenuated the lesions indicated by histology study. RT-PCR and Western blot were employed to detect the mRNA and protein expression of JAK2 and STAT3 after anti-IL-23 treatment. ELISA was utilized to measure the levels of MDA (malondialdehyde) and superoxide dismutase (SOD). Moreover, curcumin and IL-6 were implicated in the endogenous intervention of IL-23/IL-23R signaling in vivo. Our data demonstrated that the treatment of anti-IL-23 might transcriptionally activate the classic immune pathway in the brain. Anti-IL-23 augment the phosphorylation levels of both JAK2 and STAT3, suggesting the amplication signaling of JAK/STAT after exogenous IL-23 intervention. Anti-IL-23 reduced the ROS molecules of STAT downstream in the serum and brain. It also alleviated the injury by lowering the levels of MDA and SOD in the serum. JAK2 inhibitor could abolish the effect of anti-IL-23 whereas JAK3 ameliorated the injury. The combination of anti-IL-23 and JAK3 displayed synergetic results.


Results
Our data demonstrated that the treatment of anti-IL-23 might transcriptionally activate the classic immune pathway in the brain. Anti-IL-23 augment the phosphorylation levels of both JAK2 and STAT3, suggesting the ampli cation signaling of JAK/STAT after exogenous IL-23 intervention. Anti-IL-23 reduced the ROS molecules of STAT downstream in the serum and brain. It also alleviated the injury by lowering the levels of MDA and SOD in the serum. JAK2 inhibitor could abolish the effect of anti-IL-23 whereas JAK3 ameliorated the injury. The combination of anti-IL-23 and JAK3 displayed synergetic results.

Conclusions
In summary, this study indicated that anti-IL-23 had protection effects against cerebral ischemiareperfusion injury by targeting the immune speci c JAK2-STAT3 in JAK/STAT pathway.

Background
Cerebral vascular diseases, such as cerebral hemorrhage and cerebral ischemia are severe neural diseases which could result in disability and mortality 1,2 . It is known that insu cient blood supply in brain vessels could cause ischemia induced injury 3 . Multiple molecular mechanisms are involved in the ischemia induced injury caused by insu cient blood supply in brain vessels, including neural apoptosis, DNA damage, neurotransmitter release, oxidative stress and hypoxia 4 .
Accumulative evidence has implied that in ammation plays an important role in the neural damage or repairment through diverse signaling pathways 5,6 . For example, C1-inhibitor, a complement and contactkinin systems inhibitor, is neuroprotective in cerebral ischemia by decreasing the pro-in ammatory cytokines such as TNF and IL-18 and increasing the protective cytokines such as IL-6 and IL-10 gene expression 7,8 . C1-inhibitor exerted an anti-in ammatory and anti-apoptotic function on ischemiareperfusion injury which also indicates in ammation are essential for the nerve injury 9, 10 .
Neuroprotective agents derived from natural herbs with anti-in ammatory and anti-oxidant activities could alleviate neural damage nerve injury after cerebral ischemia-reperfusion injury through inhibiting apoptosis and in ammation 11,12 . Besides the chemical inhibitors or natural compound, agents such as pure proteins or targeted antibodies might contribute to the protection against cerebral ischemiareperfusion injury 13,14 .
Anti-IL-23 is an antibody to a novel immune related cytokine of IL-23. IL-23 has been demonstrated to exert pro-in ammatory functions in a series of brain diseases such as stroke 15 , aneurysmal subarachnoid hemorrhage 16 , as well as cerebral ischemia/reperfusion in ammatory injury 9 . In our preliminary experiment, we found that the proliferation and expansion of Th17 cells with potent proin ammatory cytokine IL-17 production is driven by IL-23. Moreover, IL-23 can drive the IL-17 signaling which is associated with the IL-17 producing CD8 T cells. Although IL-23 is functionally implicated in the host defense responses against acute infection and chronic autoimmune diseases, the effect of IL-23 antibody (anti-IL-23) on the neural vascular diseases remain uncertain. Here, we aimed to apply anti-IL-23 in the cerebral ischemia-reperfusion injury model to analyze its therapeutic features in the nerve damage in cerebral vascular diseases.

Rat MCAO Model
Male adult Sprague-Dawley rats (250 grams) were purchased from Sparford, China. The experiments were approved by Animal Use Committee of Beijing Chaoyang Hospital, Capital Medical University. The middle cerebral artery occlusion (MCAO) of the rats was anesthetized for 2 h, followed by 24 hours of reperfusion. We separated the right internal carotid and internal carotid arteries (ICA) and common carotid artery (CCA). Then, the right external carotid artery (ECA) and CCA were ligated to the distal and proximal ends. We inserted a piece of nylon lament from the ECA in ICA until it reached the middle cerebral artery (MCA). The diameter of thread was about 0.235 mm, and the thread was inserted from the common carotid artery to the right ICA until it passed the MCA origin (approximately 20 mm). After reperfusion for 24 hours, the rats were sacri ced, and the brains were harvested. The rats were anesthetized with 10 % chloral hydrate intraperitoneally (300 mg/kg, i.p.). The cerebral infarct volume was evaluated by using 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining. Other specimens were homogenized in ice-cold normal saline by 1:9 (w/v) for the future experiments.

Drug administration
In the control group, the rats had MCAO. Goat polyclonal anti-IL-23 antibody was purchased from Santa Cruz Biotechnology, USA. 4 μg anti-IL-23 was administered to the right lateral ventricle by a syringe when reperfusion initiated, according to previously published protocol 17 .

Measurement of infarct volume
The rats were given an overdose of chloral hydrate and decapitated. Brains were quickly removed, rinsed in cold normal saline, placed at −80°C for 3 min, and sliced into 2 mm-thick coronal sections. Tissue sections were incubated for 30 min at 37°C in a solution of 2% TTC in normal saline in the dark. The borders of the infarct in each brain slice were outlined and the area quanti ed using Image J, an NIH image software. To correct it for brain swelling, the infarct area was determined by subtracting the area of undamaged tissue in the left hemisphere from that of the intact contralateral hemisphere. The infarct volume was calculated by using the slice thickness and the measured area of the lesion. Infarct area = Contralateral hemisphere area -healthy area of ipsilateral hemisphere. Infarct volume = Infarct area × thickness of slice. Data are expressed as a percentage of the total hemisphere.

Measurement of MDA, SOD and GPX activities
The homogenized of brain tissue was centrifuged (1760 g×10 min), the supernatant was collected for measuring MDA content and activities of SOD, GPX. GPX activity was measured at 412 nm by spectrophotometer, SOD activity was measured at 550 nm by xanthine oxidase method.

RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, San Diego, CA, USA) in accordance with the manufacturer's instructions. Reverse transcription and quantitative real-time PCR using SYBR Green were performed to compare the relative expression levels of speci c mRNAs, as described previously. Real-time PCR measurements were performed to obtain a mean C T value for each sample. The values of the different samples were compared using the 2 -ΔΔCT method, and Actin expression levels were used as an internal reference.

Western blot
The cerebral tissues were homogenized in the ice-cold buffer. Then, total proteins were lysed. We performed SDS-PAGE electrophoresis and sent the proteins to polyvinylidene uoride membranes (Millipore, USA). The proteins were treated by primary antibodies. After washing, the membrane was treated by the corresponding IgG-HRP secondary antibody (1: 2000, sc-2004, Santa Cruz) for an hour.
GelDoc-2000 Imagine system was used to measure chemiluminescence to visualize and quantify signal bands. The protein level was normalized to β-actin.
Oxidative stress Malondialdehyde (MDA) is an oxidative stress marker, and superoxide dismutase (SOD) is an antioxidant enzyme. Their levels were examined which were treated as oxidative stress. SOD activities were detected via the ability to inhibit the formation of formazan dye with maximum absorbance at 450 nm. The contents of MDA were assessed at 532 nm via reacting with thiobarbituric acid to form a chromophoric product.
H&E staining Three brain sections for each case taken from the injury area were stained with H&E (Sigma-Aldrich). Brie y, the sections were warmed at 37°C overnight. After OCT was eluted with PBS for 10 minutes, the sections were stained with hematoxylin solution for 8 minutes. After differentiation in 0.5% acid alcohol for 10-15 seconds, the sections were washed in running tap water for 10 minutes, followed by dehydration in 75% and 85% alcohol for 5 minutes each. The sections were then counterstained in eosin solution for 25 seconds, quickly dipped in 95% alcohol four times, dehydrated in 95% alcohol, with two changes of absolute alcohol for 5 minutes each, followed by clearing with two changes of xylene for 5 minutes each. The sections were cover-slipped using PermountTM Mounting Medium (0909129, Shanghai Zhanyun Chemical Co., Ltd., Shanghai, China).
The slices from anterior to posterior, with left hemisphere on the left, were lined up on a piece of blue plastic. Excess water was removed on and around the slices with a paper towel. A ruler was placed alongside the slices. Photos were taken with a digital camera (Olympus BX 53 microscope, Tokyo, Japan).

TUNEL staining
Apoptotic cells were detected using a TUNEL kit (Zhongshan Bio., China). Tissues of the normal and the model groups were de-waxed and dehydrated. After washing, the tissues were treated by proteinase K in a wet chamber at 37 ° C for 30 minutes. 50 μl TUNEL was added. Transcription activator protein = was added and incubated for 30 minutes. Tissues were then stained by DAB, dehydrated, permeabilized, and sealed. Using a microscope, 5 non-overlapping areas were selected in the ischemic area, and the number of cells was measured. A uorescence microscope was utilized to record the results (LSM510META, Zeiss).
Immunohistochemistry staining CD68 and TNF-α were immunohistochemically stained. The heart tissues were xed, embedded in para n, dewaxed, and rehydrated. After blocking, tissues were treated with anti-mouse CD68 or TNF-a (Abcam, UK) at 4 ° C for a night. The tissues were treated with Chromogen (DakoCytomation, Denmark) and stained with hematoxylin (Sigma, USA). For morphology, tissues were observed by a microscope (Zeiss, Germany) x400.

Statistical analysis
Statistical analysis was performed using SPSS 17.0 software. Results represent the average of three independent experiments, and data are expressed as mean ± standard deviation (SD). Comparisons between the two groups were analyzed by Student's t-test. Multiple comparisons between groups were evaluated by one-way analysis of variance, and then Tukey's multi-range post hoc test was performed. P<0.05 was considered statistically signi cant.

Results
The effect of anti-IL-23 on the cerebral infarction induced by reperfusion The cerebral ischemia-reperfusion injury model was established using MCAO. anti-IL-23 was injected through i.p. injection. In sham group, no neurological de cits were observed. After 24 hours after reperfusion, anti-IL-23 treatment improved the neurological score compared to MCAO rats which displayed severe neural symptoms. As shown in gure 1A, MCAO showed a dramatic cerebral infarction after reperfusion, and this was prevented by the treatment of anti-IL-23 via injection with lower cerebral infarction volume. By H&E staining and TUNEL staining, less in ammatory in ltration and apoptosis were consistently observed on the anti-IL-23 treatment group ( gures 1B and 1C). In addition, the receptor of IL-23 (IL-23R) was highly expressed on the brain of MCAO rats compared with the sham group ( gure 1D), suggesting the anti-IL-23 might exhibit effect on ischemia/reperfusion via its receptor IL-23R, and its transcription pattern was further con rmed by qPCR ( gure 1E).

The effects of anti-IL-23 on the protein expressions of MDA, SOD, and GSH-Px caused by ischemiareperfusion injury
To further evaluate the effects of anti-IL-23 in cerebral ischemia injuries, we evaluated oxidative parameters like MDA and SOD. From gure 2, we found that anti-IL-23 also improved series of neural ischemia factors including MDA, SOD and GSH-Px. anti-IL-23 treatment lowered the MDA content in the MCAO ( gure 2A). In addition, anti-IL-23 treatment enhanced cerebral SOD ( gure 2B) and GSH-Px activities caused by ischemia/reperfusion on the rats ( gure 2C). Our results con rmed that anti-IL-23 lowered the oxidative stress induced by cerebral ischemia-reperfusion injury.

Anti-IL-23 activated JAK2 and JAK3
To further explore the possible mechanism of anti-IL-23 on the MCAO, we isolated the brain tissues and we found that anti-IL-23 treatment could activate the phosphorylation of JAK2 and STAT3 in vivo. In addition, anti-IL-23 exposure also enhanced the level of phosphorylated JAK3, suggesting that anti-IL-23 triggered JAK/STAT signaling might play an essential role in the immune regulation on neural damage ( gure 3A). However, anti-IL-23 did not affect the molecules' transcriptional pattern in this pathway ( gure 3B). Although anti-IL-23 is capable of activating both JAK2 and JAK3, it was observed that JAK2 inhibitor could abolish the effect of anti-IL-23 whereas JAK3 inhibitor attenuated the severity and the combined of anti-IL-23 and JAK3i displayed synergetic effect represented by infarct volume ( gure 3C).

IL-23 suppressor (curcumin) attenuated the effects of IL-23 on cerebral ischemia injury
Besides the exogenous IL-23 intervention, it was reported that curcumin repressed the high expression of interleukin-23 and interleukin-17 expression in rat retina after retinal ischemia-reperfusion injury 18 . On the contrary, here we found that curcumin treatment directly suppressed the IL-23 expression in the brain of cerebral ischemia-reperfusion using MCAO model ( gure 4A). Since IL-6 is necessary for IL-23R mRNA expression 19 , this was also con rmed in the brain tissue after IL-6 injection ( gure 4B). Consistently curcumin treatment also ameliorated the injury ( gure 4C). In addition, cerebral ischemia-reperfusion injury rats treated with curcumin plus IL-23 showed more severe symptom of neuronal impairment compared with rats which received IgG control antibody ( gure 4D). However, the injection of proin ammatory IL-17 failed to improve the infarction ( gure 4E) indicating that the effect of anti-IL-23 on cerebral ischemia-reperfusion injury did not depend on its IL-17 induction activity.

Discussion
IL-23 is a heterodimeric cytokine member of the IL-12 cytokine family, which is comprised of IL-23 speci c p19 subunit and IL-12p40 subunit, shared with IL-12 9,15 . Macrophages and dendritic cells upon activation can express p19 and p40 for the production of IL-23 20,21 . The functional IL-23 receptor complex also consists of two receptor subunits including IL-23-speci c receptor subunit (IL-23R) as well as IL-12 receptor beta 1 subunit (IL-12R beta 1) 22,23 . IL-23 and IL-12 are characterized by overlapping but distinct biological functions since IL-12 could drive Th1 type cell development whereas IL-23 is postulated as a cytokine that drives IL-17-and IL-22-producing Th17 cells development. IL-17 could also be produced by IL-23 in NK cells and neutrophils 24,25 . In addition, IL-23 appeared to be critical for IL-17Aproducing γδ T cells especially in experimental arthritis 26 . IL-23 is functionally involved in the pathological process of autoimmune diseases such as psoriasis and rheumatoid 27 . For instance, highly expressed IL-23 in psoriasis can lead to increase in numbers of Th17 cells for the pathogenesis of skin lesions 28 . Moreover, trials with IL-23p40 and p19 blockers indicate that IL-23 is a valid therapeutic target for Crohn's disease 23 .
IL-23 is pleiotropic in diverse immune cells and the dysfunction of the IL-23-IL-17 immune axis plays a crucial role in organ-speci c autoimmune in ammatory diseases 29 . However, targeting IL-23 on the cerebral ischemia-reperfusion injury remains less clear. In fact, IL-23 has been implicated in regulating neural in ammation 24,30 . IL-17-producing γδ T cells were found at high frequency in the brain of mice with experimental autoimmune encephalomyelitis (EAE), forming an ampli cation loop for IL-17 production by Th17 cells and increasing the susceptibility to EAE 31,32 . Different from its in ammatory function in EAE, we observed its neural protective effect of anti-IL-23 on the cerebral ischemia-reperfusion injury which is independent of IL-17 production in the present study. Anti-IL-23 could induce IL-17 production in brain, whereas IL-17 treatment failed to improve the diseases.
The cytosolic Janus kinases are linked with receptors on the membrane for rapid transduction of signals from the cell surface to the nucleus 33,34 . So far, four JAKs subtypes including JAK1, JAK2, JAK3 and Tyk2 have been identi ed in mammals, cytokines as the ligands for receptors cause the activation of JAK and downstream STAT (signal transducers and activators of transcription) 35,36 .
It is known that STATs are a unique class of transcription factors with seven members including STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 which are essential for regulating genes expression 34,35 . STATs are initially recruited to receptor signaling complex and phosphorylated by JAKs, forming homodimers or heterodimers which then translocate to the nucleus and bind to gamma-IFN activation site (GAS) motifs for the gene transcription 37,38 . Thus, cytokines stimulation via receptors is critical for the activation of JAK-STAT. For instance, G-CSF prevented cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes 39 . The IL-10 receptor signaling through JAK-STAT pathway was also reported 40 . Recently, more evidence also revealed that IL-17/IL-23 axis is associated with the JAK-STAT pathways 41 . Nickel Sulfate promotes Th17 cells in an IL-23-Dependent manner via Jak-STAT Pathways using speci c inhibitor (JAK inhibitor) 42 . A plausible mechanism of action of tofacitinib targeting JAKs in psoriatic arthritis (PsA) is likely to be inhibition of the IL-23/IL-17 cytokine axis via suppressing the anti-IL-23-induced JAK-STAT signaling system 43 .
In this study, we investigated the role of anti-IL-23 on cerebral ischemia-reperfusion injury using the establishment of MCAO model. However, further evidence of the mechanism of IL-23 is still required. For instance, although we detected the increasing expression of IL-23R in MCAO, the cell source of IL-23/IL-23R remains unclear. In the future, we would isolate the diverse neural cells including neurons, microglial, or in ltrated immune cells in the CNS to ascertain which cell types could express the molecules targeting IL-23 that are bene cial for the therapy of cerebral ischemia.

Conclusions
In summary, this study indicated that anti-IL-23 had protective effects against cerebral ischemiareperfusion injury by targeting the immune speci c JAK2-STAT3 in JAK/STAT pathway.

Declarations
Ethics approval and consent to participate This study was approved by animal use committee of Beijing Chaoyang Hospital, Capital Medical University and conducted in strict accordance with the national institutes of health guidelines for the care and use of experimental animals.