A20 Ameliorates Intracerebral Hemorrhage− Induced Inflammatory Injury by Regulating TRAF6 Polyubiquitination

This information is current as Zhaoyou Meng, Ting Zhao, Kai Zhou, Qi Zhong, Yanchun of September 26, 2021. Wang, Xiaoyi Xiong, Faxiang Wang, Yuanrui Yang, Wenyao Zhu, Juan Liu, Maofan Liao, Lirong Wu, Chunmei Duan, Jie Li, Qiuwen Gong, Liang Liu, Ao Xiong, Meihua Yang, Jian Wang and Qingwu Yang

J Immunol published online 16 December 2016 Downloaded from http://www.jimmunol.org/content/early/2016/12/15/jimmun ol.1600334

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 16, 2016, doi:10.4049/jimmunol.1600334 The Journal of Immunology

A20 Ameliorates Intracerebral Hemorrhage–Induced Inflammatory Injury by Regulating TRAF6 Polyubiquitination

Zhaoyou Meng,* Ting Zhao,* Kai Zhou,* Qi Zhong,* Yanchun Wang,* Xiaoyi Xiong,* Faxiang Wang,* Yuanrui Yang,* Wenyao Zhu,* Juan Liu,* Maofan Liao,* Lirong Wu,* Chunmei Duan,* Jie Li,* Qiuwen Gong,* Liang Liu,* Ao Xiong,† Meihua Yang,* Jian Wang,‡ and Qingwu Yang*

Reducing excessive inflammation is beneficial for the recovery from intracerebral hemorrhage (ICH). Here, the roles and mechanisms of A20 (TNFAIP3), an important endogenous anti-inflammatory factor, are examined in ICH. A20 expression in Downloaded from the PBMCs of ICH patients and an ICH mouse model was detected, and the correlation between A20 expression and neurologic deficits was analyzed. A20 expression was increased in PBMCs and was negatively related to the modified Rankin Scale score. A20 expression was also increased in mouse perihematomal tissues. A202/2 and A20-overexpressing mice were generated to further analyze A20 function. Compared with wild-type (WT) mice, A202/2 and A20-overexpressing mice showed significant increases and decreases, respectively, in hematoma volume, neurologic deficit score, mortality, neuronal degeneration, and proinflammatory factors. Moreover, WT-A202/2 parabiosis was established to explore the role of A20 in peripheral blood in ICH injury. ICH- http://www.jimmunol.org/ induced damage, including brain edema, neurologic deficit score, proinflammatory factors, and neuronal apoptosis, was reduced in A202/2 parabionts compared with A202/2 mice. Finally, the interactions between TRAF6 and Ubc13 and UbcH5c were increased in A202/2 mice compared with WT mice; the opposite occurred in A20-overexpressing mice. Enhanced IkBa degra- dation and NF-kB activation were observed in A202/2 mice, but the results were reversed in A20-overexpressing mice. These results suggested that A20 is involved in regulating ICH-induced inflammatory injury in both the central and peripheral system and that A20 reduces ICH-induced inflammation by regulating TRAF6 polyubiquitination. Targeting A20 may thus be a prom- ising therapeutic strategy for ICH. The Journal of Immunology, 2017, 198: 000–000.

ntracerebral hemorrhage (ICH), which results in high mor- and the release of proinflammatory factors all participate in the by guest on September 26, 2021 tality and morbidity, accounts for 10–15% of all stroke cases; pathogenesis of inflammation (4, 5). Most previous studies have I mortality is especially high in China, but effective therapies focused on the role of inflammatory factors. As a result, endog- remain limited (1–3). Previous studies have indicated that in- enous anti-inflammatory factors that can ameliorate ICH-induced flammation plays a key role in ICH-induced secondary injury and inflammation have been overlooked (6–8). Thus, targeting en- that microglia activation, peripheral inflammatory cell infiltration, dogenous anti-inflammatory factors could provide insights into the development of new ICH therapies. Zinc finger protein A20 (TNFAIP3) is an endogenous anti- *Department of Neurology, Xinqiao Hospital, Third Military Medical University, inflammatory factor that can reduce the expression of destruc- † Chongqing 400037, China; Basic Medical College, Zhengzhou University, Zhengzhou tive proinflammatory factors such as IL-1b and TNF-a by 450000, China; and ‡Department of Anesthesiology and Critical Care Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21205 inhibiting NF-kB activation (9, 10). A20 also interacts with IL-10 ORCID: 0000-0001-8540-6488 (Q.Y.). and TGF-b and reduces inflammation in diabetes and arthritis (11–13). Evidence has shown that A20 is essential for restricting Received for publication February 29, 2016. Accepted for publication November 15, 2016. TLR-induced TNF receptor–associated factor (TRAF) 6 ubiq- This work was supported by grants from the National Natural Science Fund for uitination (11, 14). Our previous studies have shown that TLR4/ Distinguished Young Scholars (Grant 81525008) and the National Basic Research NF-kB signaling plays an important role in ICH-induced in- Program of China (Grant 973 Program and Grant 2014CB541605). flammatory injury (15, 16). Thus, we hypothesized that A20 may Address correspondence and reprint requests to Prof. Qingwu Yang, Department of also ameliorate ICH-induced inflammation by negatively modu- Neurology, Xinqiao Hospital, Third Military Medical University, No. 183, Xinqiao Main Street, Shapingba District, Chongqing 400037, China. E-mail address: lating TLR signaling. [email protected] The present study aimed to investigate two hypotheses: 1) A20 The online version of this article contains supplemental material. can ameliorate morphological (hematoma and brain edema) and Abbreviations used in this article: ACHBLF, acute-on-chronic hepatitis B liver fail- behavioral outcomes after experimental ICH in mice; and 2) ure; Co-IP, coimmunoprecipitation; ICH, intracerebral hemorrhage; LV, lentiviral; A20 can reduce NF-kB activation by regulating TRAF6 poly- mRS, modified Rankin Scale; NDS, neurologic deficit score; qRT-PCR, quantitative real-time PCR; RNF11, ring finger protein 11; shRNA, short hairpin RNA; SLE, ubiquitination. systemic lupus erythematosus; TAX1BP1, Tax1-binding protein; TNFAIP3, zinc fin- ger protein A20; TRAF, TNF receptor–associated factor; WT, wild-type. Materials and Methods This article is distributed under The American Association of Immunologists, Inc., Clinical study Reuse Terms and Conditions for Author Choice articles. To evaluate the relationship between A20 expression and neurologic def- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 icits, we screened 34 patients with ICH and 34 healthy controls for vari-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600334 2 ANTI-INFLAMMATORY ROLE OF A20 IN INTRACEREBRAL HEMORRHAGE ations in A20 expression. A total of 49 patients with ICH were identified sapiens full open reading frame TNFAIP3 sequence. A cDNA clone of between December 2013 and June 2015. The inclusion criteria were as A20 was transcribed, and the product was amplified using primers with follows: 1) identification of patients with primary ICH within 24 h of disease flanking NheI and ASCI restriction sites. The DNA was then inserted into onset and the extraction of blood from the cubital fossa vein before the pLenti6.3_MCS_IRES2-EGFP. The primer sequences are listed in Table II. administration of any drugs; 2) hemorrhage in the basal ganglia confirmed The A20 recombinant vector was named LV-A20, and the titer of the virus by computerized axial tomography; and 3) diagnosis according to the was 1.2 3 108 TU/ml. LV-A20 virus solution (3 ml) was injected into the criteria of the Fourth Chinese National Meeting for Cerebrovascular Disease lateral ventricles (0.3 mm posterior and 1 mm lateral of bregma and at a (1996) and the European Stroke Initiative (17). The exclusion criteria were depth of 2.75 mm) of the mice using a 5-ml syringe pump (KD Scientific). as follows: 1) patient age ,18 or .80 y; 2) surgical history within the last The needle was held in place for 20 min, and the microinjector was then 6 mo; 3) coma or death within 48 h after admission; 4) hematoma caused drawn out slowly. The skull was sealed with bone wax, and the scalp was by trauma, drug abuse, brain tumor, vascular malformations, anticoagulation sutured. A20 expression was detected by quantitative real-time PCR (qRT- therapy, or coagulation abnormalities; 5) obvious inflammatory disease (e.g., PCR), Western blotting, and immunofluorescence. infectious disease, systemic lupus erythematosus (SLE), or rheumatoid ar- thritis; 6) presence of a hospital-acquired infection; 7) acute and chronic Lentiviral short hairpin RNAs infection hepatopathy; 8) diabetes diagnosis; and 9) lack of agreement with the study protocol or inability to undergo all of the tests required by the study. A total Recombinant lentiviral (LV) short hairpin RNAs (shRNAs) targeted of 15 patients were excluded from the study, including 3 patients who died to mouse TRAF6 (Gene ID: 22034, named LV-TRAF6 shRNA) 9 9 and 12 who were lost to follow-up. A total of 34 patients were enrolled in the were designed (shRNA1: 5 -GCAAGTATGAGTGTCCCATCT-3 ; shRNA2: 59-GCTGTCCTCTGGCAAATATCA-39; shRNA3: 59-GGAG- study (for general clinical characteristics of ICH patients and the healthy GACAAGGTTGCCGAAAT-39) by Shanghai GenePharma (China). control group, see Table I). Upon admission to our hospital, 5 ml cubital vein 9 blood was extracted for A20 detection using EDTA as an anticoagulant Negative-control LV particles were also designed (5 -GTCTTCGAACG- 9 agent. PBMCs were isolated using a PBMC kit, according to the manufac- TATCAAGT-3 ) and packaged. The final titers of LV-TRAF6 shRNA and negative-control LV particles were 2 3 108 TU/ml. A total of 5 ml of virus turer’s instructions (10771; Sigma), to determine A20 expression. Modified Downloaded from Rankin Scale (mRS) scores were used to evaluate neurologic deficits in the solution was injected into the lateral ventricles of mice using a micro- injector. TRAF6 knockdown efficiency was confirmed by Western blotting patients 3 mo after ICH (18). Correlation analysis was used to analyze the (see Supplemental Fig. 2A). LV-TRAF6 shRNA1 showed the best relationship between A20 mRNA expression and the patient mRS scores. All blood samples collected from the healthy controls and patients were used knockdown effect, and was therefore selected for lateral ventricle injec- with their informed consent, and the study procedures were approved by the tion. Then, 7 d after LV-TRAF6 shRNA injection, the mice underwent Ethics Committee of Xinqiao Hospital of the Third Military Medical Uni- surgery for the ICH model. Subsequently, we harvested the brain tissues k versity, China, and conducted in accordance with the Declaration of Helsinki and tested NF- B expression and downstream proinflammatory cytokine and its amendments. release by Western blotting and ELISA, respectively. http://www.jimmunol.org/ Animals Neurological deficit scores C57BL/6 mice (male, 8-wk-old, 18–22 g) were obtained from the Animal The experiment was conducted according to our previous methods (16). A Center of the Third Military Medical University (Chongqing, China). 28-point neurologic deficit score (NDS) assessment system was adopted. A202/2 mice were purchased from Riken BioResource Center, Japan. The Climbing, front limb symmetry, circling behavior, and body symmetry C57BL/6 genetic background was maintained over the course of six were assessed. The scoring was completed by two blinded laboratory re- generations of hybridization in the A202/2 mice, and these mice were searchers who were unaware of the group assignments of the mice. The average score was used as the final score for each mouse. identified using PCR. All mice were raised in specific pathogen-free–grade animal rooms in a temperature- and light-controlled environment. The Hematoma measurement by guest on September 26, 2021 mice were also provided with unlimited access to food and water. All procedures were approved by the Animal Management Committee of the The experiment was conducted according to our previous methods (15). Third Military Medical University, China. Briefly, mice were anesthetized with a lethal dose of chloral hydrate, then perfused and fixed. The brains were then removed and sectioned from the Parabiosis frontal lobe to the occipital lobe to prepare continuous 40-mm-thick co- ronal sections. One out of every five sections was collected, and the fixed Parabiosis was developed as described previously (19, 20). Briefly, the mice brain slices were arranged in order. Image-Pro Plus 5.0 image processing were anesthetized by i.p. injection with Zoletil (25 mg/kg containing 0.2% Rompun). The mice were then shaved and a unilateral flank skin incision software (Media Cybernetics, Bethesda, MD) was used to measure the V t 3 A ... from the elbow to the knee joint was created. The skin edge of the mice hematoma volume (microliter ) according to the equation = ( 1+ + A V t A was sutured with 5.0 prolene (Ethicon). The suture area was daubed with n), where is the hematoma volume, is the slice thickness, and i is the erythromycin ointment, and the partners were fixed with gauze to avoid area of bleeding. Hemoglobin content in the hematoma tissues was also tearing the wound. The wild-type (WT) mouse was used as the donor measured to further quantify hematoma size (19). Blood (0, 2, 4, 8, 16, and 2/2 20 ml) was added to fresh brain homogenates, and the OD values of the parabiont and the A20 mouse as the recipient partner. At the same time, samples were measured at 540 nm using a spectrophotometer (Thermo WT-WT parabionts were constructed as controls. ICH was induced in the WT or A202/2 parabionts 20 d after parabiotic surgery. Multiskan, Pittsburgh, PA). These values were then used to plot a standard curve. The striatum of mice in each group was removed 3 d after ICH and ICH model dissolved in Drabkin’s reagent. The supernatant of the homogenates was collected, and the OD was measured using a spectrophotometer; the he- The ICH model was induced according to our previously described pro- matoma volume was then calculated using the standard curve. cedure (16). Whole blood (20 ml) without anticoagulant or the same vol- ume of saline was injected at 2 ml/min into the right striatum (0.8 mm Brain water content anterior and 2 mm lateral of bregma and at a depth of 3.5 mm) using a syringe pump (KD Scientific, Holliston, MA). The success rate of the ICH As described in our previous report (16), mice were randomly selected from model was 90%; failed models and dead mice were excluded from the eachgroupat1,3,and7dafterICHtodetectthebrainwatercontent.Briefly, experiment. the cerebral tissues were removed after the mice were anesthetized with chloral hydrate, and then the samples were divided into five parts: the ipsilateral Survival analysis cortex, the ipsilateral basal ganglia, the contralateral cortex, the contralateral basal ganglia, and the cerebellum. The brain water content was measured using The number of dead mice was counted at 1, 3, 5, and 7 d after ICH, and the the following formula: [(wet weight – dry weight)/wet weight] 3 100. survival rate was calculated as follows: (the number of ICH mice per group – the number of dead mice per group)/the number of ICH mice per group. Quantitative real-time PCR Kaplan–Meier survival plots were produced using a log-rank test in GraphPad Prism 5. Significance was set at p , 0.05. Total RNA was extracted from the tissue using TRIzol reagent (Invitrogen, Gaithersburg, MD), and qRT-PCR was performed according to the man- Construction of the A20 overexpression mouse model ufacturer’s instructions (Takara Biotechnology, Dalian, China). GAPDH was used as an internal control. The primers used in the study are listed in Table II. PCR primers for A20 (NM_001270508.1) and the lentiviral vector Relative levels of mRNA expression were calculated using the 22OOCT (pLenti6.3_MCS_IRES2-EGFP) were designed according to the Homo method. The Journal of Immunology 3

Western blotting immunofluorescence. The baseline level (e.g., the age, male sex, Proteins from perihematomal tissues were resolved by SDS-PAGE and body temperature, history of vascular risk factors, laboratory transferred onto polyvinylidene fluoride membranes by electroblotting (16). parameters, obvious inflammatory disease within 6 mo, acute The membranes were incubated overnight with a rabbit anti-mouse myocardial infarction, and acute or chronic liver damage) was TNFAIP3 Ab (1:200, ab13597; Abcam) at 4˚C. GAPDH was used as a comparable across both groups (Table I). A20 expression in the loading control, and membranes were incubated with HRP-conjugated ICH patients was significantly increased compared with that in the goat anti-rabbit secondary Abs (1:200; Millipore) at 25˚C for 1.5 h. , Bound Abs were visualized using a chemiluminescence detection system. healthy controls (p 0.01) (Fig. 1A, 1C). Correlation analysis Signals were quantified by scanning densitometry and computer-assisted indicated that A20 expression was negatively correlated with pa- image analysis. Protein levels were expressed as the ratio of the value of tient mRS scores 3 mo after ICH (R2 = 0.6257) (Fig. 1B). the detected protein band to the GAPDH band. A20 expression increased after ICH in mice, especially in Immunofluorescence microglia and neurons Following our previously described method (21), mouse brain tissues were To further investigate the role of A20, an ICH mouse model was fixed in 4% paraformaldehyde. The samples were subjected to gradient dehydration and were then embedded, frozen, and cut into 5-mm-thick used to analyze A20 expression in perihematomal tissue. qRT-PCR sections. A20 expression was then detected using double fluorescence results indicated that compared with the sham group, A20 ex- immunohistochemistry. The primary Abs used in this experiment are listed pression increased at 12 h after ICH, peaked at 1 d and then in Table III. Positive cells were calculated and analyzed in three different gradually declined (Fig. 2A). Western blotting indicated that A20 arbitrary units that can be defined as the average number of positive cells in expression increased at 12 h, peaked at 3 d, and then gradually three randomly selected fields (8003) in three slices of each experiment group. declined. Seven days after ICH, A20 expression in the model was Downloaded from still higher than in the sham group (Fig. 2B). Because A20 ex- Cytokine production pression peaked at 3 d after ICH, we chose this time point to Brain samples were collected at the indicated times and assayed using a further analyze the A20-expressing cells. The results indicated mouse IL-1b, IL-6, and TNF-a ELISA Kit (Dakewe, Beijing, China). that A20 expression was more prominent in microglia and neurons Apoptosis assay than in astrocytes (Fig. 2C). http://www.jimmunol.org/ A TUNEL detection kit (12156792910; Roche, Germany) was used to A20 knockout increases inflammatory injury in perihematomal evaluate neuronal apoptosis. Cells with blue-black nuclei were considered tissue after ICH TUNEL-positive. For each group, the number of positive cells in three randomly chosen high-power fields (4003) within the perihematomal area Because A20 expression in the brain was significantly increased 3 d was counted using a light microscope. after ICH, inflammatory injury was analyzed in the perihematomal tissue. Brain edema and NDS are important indexes for assessing Immunoprecipitation ICH-injury severity. In this experiment, hematoma volume and Immunoprecipitation was performed as described previously (16). Peri- hemoglobin content in the A202/2 mice were increased compared hematomal tissues were collected from the mice and solubilized on ice in with those in the WT mice (Fig. 3A). Compared with WT mice, radioimmunoprecipitation assay lysis buffer. All lysates were then sub- 2/2 jected to SDS-PAGE, transferred to nitrocellulose membranes, and blocked the water content in the ICH ipsilateral hemisphere of the A20 by guest on September 26, 2021 with 5% skim milk. All extracted proteins were incubated with specific mice was higher at 3 d after ICH (Fig. 3B). Here, we observed primary and secondary Abs or with IgG as a control (Millipore). The NDS deterioration in mice after A20 deficiency compared with the resulting immunoprecipitates were centrifuged and resuspended in SDS- WT group at all time points (Fig. 3C). Furthermore, we assessed PAGE buffer. Protein expression was measured by Western blotting. The Abs used in this experiment are listed in Table IV. changes in the expression of inflammatory factors, including IL- 1b, TNF-a, and IL-6, in the perihematoma tissue of mice after EMSA ICH using the ELISA. The results showed that IL-b, IL-6, and At 1, 3, and 7 d after ICH, the mice from each group were sacrificed TNF-a expression in the WT ICH mice was elevated compared and their brains removed. Nuclear proteins were extracted from peri- with that in the WT sham group, and the expression levels in the hematomal brain tissue as described previously (22). Equal amounts of A202/2 ICH mice were even higher than those in the WT ICH protein were incubated for 15 min at room temperature with an NF-kB– 32 mice (Fig. 3D). The death rate was also significantly increased specific [ P]-labeled oligonucleotide and binding mix following the kit 2/2 instructions (GS056B; Beyotime, Nantong, China; LightShift Chemilu- after ICH in the A20 mice (Fig. 3E). Neuronal apoptosis was minescent EMSA Kit; Pierce). Competition assays were conducted by also detected by TUNEL staining, and compared with WT mice, adding a 100-fold excess of unlabeled NF-kB oligonucleotide to the the A202/2 mice exhibited an increase in TUNEL-positive cells in reaction mix. For supershift experiments, Abs were preincubated with the perihematomal area (Fig. 3F). Thus, A20 can protect the brain thesample(fromA202/2 mice 3 d after ICH) for 10 min prior to in- cubation with radiolabeled probe. The images were analyzed on a Bio- from inflammatory injury after ICH. These results indicated that Rad image analyzer. A20 deficiency significantly aggravated ICH-induced inflamma- tory injury in a mouse model of ICH. Statistical analysis Data are expressed as the mean 6 SEM or as percentages. Statistical A20 overexpression can reduce post-ICH inflammatory injury differences between pairs of groups were assessed using the unpaired t test in mice and between multiple groups using two-way ANOVA. Correlation analysis was used to analyze the relationship between A20 mRNA expression and To further evaluate the role of A20 in ICH-induced inflammatory the mRS scores of the ICH patients. Differences were considered signifi- injury in mice and to evaluate the therapeutic effect of A20 on ICH, cant at p , 0.05. we developed a mouse model of A20 overexpression. The qRT- PCR and Western blotting showed that A20 expression was higher 7 d after LV-A20 injection compared with the sham and Results vector groups (Fig. 4A, a1, a2). Moreover, we performed immu- A20 expression was increased in the PBMCs of ICH patients nofluorescence to examine A20 expression in different cell types and negatively correlated with neurologic deficits in brain 7 d after LV-A20 injection. A20 was mainly expressed in The expression of A20 in PBMCs from 34 ICH patients and 34 sex- microglia and neurons (Supplemental Fig. 1), which is consistent and age-matched healthy controls was analyzed by qRT-PCR and with the cell types showing A20 expression after ICH (Fig. 2C). 4 ANTI-INFLAMMATORY ROLE OF A20 IN INTRACEREBRAL HEMORRHAGE

Table I. General clinical characteristics of ICH patients and healthy control group

Control (from MEC) ICH Patients p Age, y 59.8 (9.7) 62.1 (10.82) 0.62 Male, % 56.4 59.2 0.81 Body temperature, ˚C 36.9 (0.60) 37.1 (0.61) 0.59 History of vascular risk factors, % Hypertension 88.3 95.4 0.41 Alcohol consumption 34.9 40.1 0.65 Smoking habit (current) 32.6 36.8 0.67 Diabetes No No Laboratory parameters Leukocyte count, 3109/l 7.23 (1.74) 7.57 (1.36) 0.563 Serum glucose, mmol/l 6.12 (2.18) 6.47 (1.76) 0.661 Platelet count, 3109/l 189 (46) 221 (34) 0.788 Obvious inflammatory disease within 6 mo No No Acute myocardial infarction No No Acute or chronic liver damage No No IL-6, pg/ml 8.26 (3.5) 192.4 (47.3) ,0.01 TNF-a, pg/ml 28.6 (8.7) 103.2 (20.3) ,0.01

Values are presented as proportions, mean (SEM). Downloaded from MEC, medical examination center.

Thus, ICH was induced 7 d after the mice were injected with 3 ml system. Interestingly, the hematoma volume, hemoglobin content, of LV-A20 virus. At 3 d after ICH, hematoma volume and he- brain water content, inflammation factors expression, and neuro- moglobin content in the A20-overexpressing mice were signifi- nal apoptosis were all obviously decreased in the A202/2 para- cantly lower than those in the WT mice (Fig. 4B). The brain water biont group compared with the A202/2 group. These results http://www.jimmunol.org/ content in the ICH ipsilateral hemisphere of the LV-A20 mice was suggest that A20 in peripheral blood is also involved in the reg- decreased at 3 d after ICH compared with the WT and LV-GFP ulation of inflammation after ICH (Fig. 5B–E). However, the he- groups (Fig. 4C). NDS and IL-b, IL-6, and TNF-a expression matoma volume (Fig. 5B), brain water content (Fig. 5C), and were lower in the A20-overexpressing mice than in the WT mice IL-1b and TNF-a levels in perihematomal tissue still were higher (Fig. 4D, 4E). Unexpectedly, the death rate in the A20-overexpressing 3 d after ICH compared with the WT and WT parabiont groups. group remained unchanged compared with that in the WT group Thus, A20 in the peripheral system may be only partially involved (Fig. 4F). However, TUNEL staining demonstrated that A20 in the regulation of inflammatory injury after ICH. overexpression prevented ICH-induced apoptosis 3 d after ICH. A20 suppressed ICH-induced inflammatory injury by by guest on September 26, 2021 Compared with the WT and LV-GFP groups, the number of regulating the polyubiquitination of the E3 ubiquitin ligase TUNEL-positive cells was significantly decreased in the LV-A20 TRAF6 group (Fig. 4G). Thus, intracranial A20 overexpression can re- duce inflammatory damage after ICH. TRAF6 is an important transduction molecule downstream of TLR, IL-1bR, and other classical inflammatory pathways whose poly- A20 in peripheral blood may also be involved in regulating ubiquitination can induce NF-kB activity and the subsequent se- ICH-induced inflammation cretion of inflammatory factors. These results suggest that TRAF6 The above findings raise a critical question of whether A20 in the may play an important role in regulating ICH-induced inflam- peripheral system has any impact on the regulation of inflammatory mation. Using Western blotting and ELISA experiments, we found injury after ICH. We therefore used WT-A202/2 parabiosis to test that TRAF6 expression was obviously increased after ICH, and the efficacy of peripheral A20 in alleviating ICH-induced injury in the expression trend was consistent with NF-kB p65 expression the brains of A202/2 parabionts. After parabiosis between A202/2 (Fig. 6). Inhibiting the expression of TRAF6 using TRAF6- and WT mice, the circulation of the parabionts was connected targeted shRNA relieved the cerebral edema and neurologic def- so that A20 in the blood of the parabiotic transgenic mice icits, clearly downregulated NF-kB expression, and inhibited the could be transported into the A202/2 mice. Thus, the A202/2 subsequent secretion of inflammatory cytokines (Supplemental mice acquired an additional peripheral system from WT mice. Fig. 2). These results showed that inhibiting the expression of Immunofluorescence staining revealed that the same amount of TRAF6 could inhibit inflammatory injury after ICH. A20-positive cells in A202/2 parabiont PBMCs 3 d after ICH A20 can inhibit NF-kB activation by suppressing its poly- compared with WT mice and WT parabionts. However, A20- ubiquitination and hence the activation of the E3 ubiquitin ligase positive cells were not detected in the PBMCs of A202/2 mice TRAF6 (14). However, whether A20 inhibited TRAF6 poly- 3 d after ICH (Fig. 5A). This model provided a reliable approach ubiquitination via a similar mechanism in ICH-induced inflam- to test the anti-inflammatory effect of A20 in the peripheral mation remained unclear. To investigate the mechanism of TRAF6

Table II. Primers used for quantitative real-time PCR

Primers Forward Reverse Human TNFAIP3 59-TCTGCAGTCTTCGTGGCG-39 59-GCCATTGTGCTCTCCAACAC-39 Mouse TNFAIP3 59-GACCCTGAAGGACAGTGGAC-39 59-TTTGCAGCGTTGATCAGGTG-39 A20 recombinant 59-ATCATCGCTAGCGCCACCATGGCT- 59-ATCATCGGCGCGCCTTAGCCA- GAACAAGTCCTTCCTCAGGC-39 TACATCTGCTTGAACTGAAAG-39 The Journal of Immunology 5

Table III. Abs used for immunofluorescence injury. We also found that the anti-inflammatory effect of A20 in ICH may be mediated through the disruption of ubiquitin enzyme Ab Catalog No. Dilution Company complexes. To our knowledge, this study provides the first dem- Mouse anti-mouse ab13597 1:200 Abcam onstration that A20 has a protective effect in ICH and offers new TNFAIP3 insights for the development of ICH therapies. Rabbit anti-mouse GFAP ab7260 1:200 Abcam A20 in PBMCs plays a key role in inflammatory disease, but A20 Rabbit anti-mouse Iba1 019-19741 1:200 Wako expression in PBMCs differs across diseases. Increased A20 ex- Rabbit anti-mouse NeuN ABN78 1:200 Millipore Donkey anti-mouse IgG A21202 1:800 Invitrogen pression has been demonstrated in the PBMCs of patients with Alexa Fluor 488 acute lymphoblastic leukemia and acute-on-chronic hepatitis B Donkey anti-rabbit IgG A31573 1:800 Invitrogen liver failure (ACHBLF) (23, 24), whereas decreased A20 levels Alexa Fluor 647 have been observed in the PBMCs of patients with chronic in- Rabbit anti-mouse EGFP mAb#2956 1:100 CST flammatory diseases such as type-2 diabetes and SLE (12, 25). DAPI D3571 0.5 mg/ml Invitrogen However, the protective role of A20 is widely accepted, and its GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei; Iba, ionized calcium- diverse expression in different diseases does not seem to be rel- binding adaptor molecule. evant to this role. In accordance with the results of patients with acute lymphoblastic leukemia and ACHBLF, A20 expression in regulation by A20, we first examined TRAF6 protein-protein in- PBMCs increased within 24 h after ICH. The reasons for the di- teractions in ICH-induced mice using coimmunoprecipitation (Co- vergence in A20 expression remain unclear, although different

IP). TRAF6-Ubc13 and TRAF6-UbcH5c (Ubc13 and UbcH5c are stages of inflammation might cause this discrepancy. In the acute Downloaded from E2 ubiquitin-conjugating enzymes that are regarded as key me- stage of ACHBLF and ICH, increased A20 expression could in- diators of ubiquitin chain assembly) interactions were increased in 2/2 hibit inflammation, but in type-2 diabetes and SLE, due to the A20 mice compared with WT mice. In contrast, these inter- state of continuous inflammation, the consumption of A20 by actions were reduced in the A20 overexpression group compared sustaining and redundant cytokines surpasses its production, with the WT group. We also evaluated A20-binding proteins, resulting in a decrease in A20 expression. The specific mechanism which act cooperatively to suppress NF-kB activation, including requires further study. In this study, we established a parabiosis http://www.jimmunol.org/ the E3 ubiquitin ligase Itch, as well as ring finger protein 11 2/2 model to investigate the role of peripheral A20 in neuro- (RNF11) and Tax1-binding protein (TAX1BP1). In A20 mice, inflammation after ICH based on a previously reported method. TRAF6 did not interact with Itch, RNF11, or TAX1BP1. However, We observed the same amount of A20-positive cells in the PBMCs these interactions were increased in the A20-overexpressing mice 2/2 2/2 of WT-A20 parabionts as in the WT and WT parabiont groups. (Fig. 6A). In A20 mice, Ubc13 and UbcH5c expression was However, A20-positive cells were not detected among the PBMCs increased, whereas RNF11 and TAX1BP1 expression was de- in the A202/2 group. Moreover, we found that brain cytokines, creased. In the LV-A20 group, Ubc13 and UbcH5c expression was neuronal necrosis, etc., were improved in WT-A202/2 parabionts decreased, whereas RNF11 and TAX1BP1 expression was in- 2/2 2/2 2/2 than in A20 mice (Fig. 5). The number of A20 hemato- creased. No change in Itch expression was observed in the A20 poietic cells and A20-expressing hematopoietic cells were iden- by guest on September 26, 2021 or LV-A20 groups. Moreover, we detected changes in the level of tical in both WT-A202/2 parabionts and WT-WT parabionts at 3 d ubiquitinated TRAF6 at 3 d after ICH in the A20-positive, nega- after ICH, and the baseline inflammatory levels were also similar tive, and overexpression groups using Co-IP experiments. The in the brain tissue in both groups; therefore, although the A202/2 results showed that inhibiting A20 promoted TRAF6 ubiq- hematopoietic cells may secrete inflammatory mediators to over- uitination, whereas overexpressing A20 downregulated TRAF6 come the noninflammatory influence of the A20-expressing WT ubiquitination levels (Supplemental Fig. 3). Additionally, we ex- hematopoietic cells, the identical effects and baseline levels in amined the expression of IkBa and NF-kB p65 and found that, as both mice are unlikely to influence our conclusion regarding the expected, IkBa and NF-kB p65 degradation was increased in role of peripheral A20 in reducing brain injury after ICH. Based A202/2 mice, and an increase in NF-kB activation was also de- 2/2 on these findings, we postulate that peripheral systematic A20 tected in A20 group; however, the IkBa degradation, NF-kB may be partially involved in the regulation of inflammatory injury p65, and NF-kB activity were reversed in the LV-A20 group (Fig. after ICH. In this experiment, we did not separate the parabionts 6B, Supplemental Fig. 4). No binding was observed when im- after 20 d of parabiosis and then continue to construct the ICH munoprecipitations were performed with a control rabbit Ig Ab model, and so we could not calculate the mortality and NDS of the (Fig. 6C). Our results illustrate that the inhibition of NF-kB sig- mice after ICH; hence, we did not provide these data in this part of naling by A20 plays a key role in ICH by disrupting the E2 and E3 our manuscript. ubiquitin enzyme complexes.

Discussion Table IV. Abs used for Co-IP (Western blot) Inflammation plays an important role in ICH-induced secondary injury, and the current study showed that the endogenous anti- Ab Catalog No. Dilution Company inflammatory factor A20 can protect the brain from ICH-induced Mouse anti-mouse TNFAIP3 ab13597 1:200 Abcam inflammatory injury. Briefly, A20 expression was upregulated in Mouse anti-mouse UBC13 sc-376470 1:200 Santa Cruz the PBMCs of ICH patients and was correlated with the prognosis of Rabbit anti-mouse RNF11 ab154831 1:2000 Abcam 2 2 ICH, as indicated by mRS score. Furthermore, A20 / and A20- TRAF6 ab22049 1 mg/ml Abcam overexpressing mice were created, and the results from these mice Rabbit anti-mouse UBE2D3 ab176568 1:1000 Abcam also indicated that A20 can accelerate hematoma absorption, inhibit Itch sc-25625 1:200 Santa Cruz TAX1BP1 sc-15274 1:200 Santa Cruz the release of proinflammatory factors, reduce neuron degeneration, IkBa ab32518 1:2500 Abcam 2/2 and decrease neurologic deficits. Meanwhile, WT-A20 para- NF-kB p65 sc-8008 1:200 Santa Cruz biosis experiments indicated that A20 in PBMCs may be par- Normal rabbit IgG 12-370 1:200 Millipore tially involved in the regulation of ICH-induced inflammatory K63-Ub Ab179434 1:200 Abcam 6 ANTI-INFLAMMATORY ROLE OF A20 IN INTRACEREBRAL HEMORRHAGE

FIGURE 1. A20 expression was increased in the PBMCs of patients and negatively correlated with neurologic deficits. (A) A20 expression in patient PBMCs was analyzed by qRT-PCR. (B) The relationship between A20 mRNA expres- sion and the mRS scores of ICH patients 3 mo after ICH was analyzed by correlation analysis (R2 = 0.6257, p , 0.01). (C) A20 expression was analyzed by immunofluorescence (horizontal lines indicate median values). Scale bars, 25 mm. **p , 0.01 compared with healthy controls, HC. Scale bars, 25 mm. Downloaded from http://www.jimmunol.org/

The results of the current study indicated that A20 is primarily autoimmune encephalomyelitis models and LPS-induced inflam- expressed in neurons in the normal human brain and that more A20 matory models, A20 is expressed in microglia and astrocytes and was expressed in the pons, striatum, hippocampus, and medulla suppresses inflammation (28, 29). However, our results indicated compared with the frontal cortex. A20 mRNA was also detected in that after ICH, A20 is mainly expressed in microglia and neurons the cortex and hippocampus of WT mice (26, 27). In experimental and seldom in astrocytes, consistent with the cell types that were by guest on September 26, 2021

FIGURE 2. In mice, A20 expres- sion increased after ICH, especially in neurons and microglia. A20 ex- pression in the perihematomal area was detected by (A) qRT-PCR and (B) Western blotting (**p , 0.01 versus sham, n = 6). (C) A20 ex- pression in perihematomal tissue was detected by immunofluorescence (arrows indicate A20-positive cells; scale bars, 25 mm). The percent of positive A20/NeuN, A20/Microglia and A20/Astrocytes in three ran- domly chosen fields within the peri- hematomal area was counted. The data are presented as the mean 6 SEM. *p , 0.05 versus A20/Astrocytes, #p , 0.05 versus A20/NeuN. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. A20 knockout increased inflammatory injury in perihematomal tissue after ICH. (A) Representative coronal sections from WT and A202/2 mice (a1). The hematoma volume (a2) and hemoglobin content (a3) were calculated (*p , 0.05, **p , 0.01 versus WT; n = 6 each). (B) Brain water content at sham and 3 d after ICH was compared between the WT and A202/2 group. (*p , 0.05, **p , 0.01 versus WT; n = 6 each). (C) NDS at 1, 3, 5, and 7 d after ICH was compared between the WT and A202/2 groups (**p , 0.01 versus WT; n = 6, each). (D) An ELISA Kit was used to determine IL- 1b, IL-6, and TNF-a expression in WT and A202/2 mice (*p , 0.05, **p , 0.01 versus WT; n = 3 each). (E) Overall survival in the A20-deficient group was significantly lower than that in the WT group (**p , 0.01 versus WT; n = 20 each). (F) Representative TUNEL staining in WT and A202/2 mice. Scale bars, 100 mm. The number of TUNEL-positive cells was calculated (**p , 0.01, n = 3 each).

induced to express A20 after LV-A20 injection. In the current Inflammation plays an important role in ICH-induced secondary study, we found that the NDS was significantly decreased in the injury. We previously showed that inflammatory injury can be A20 overexpression group compared with the WT group after ICH induced by the activation of TLR2/TLR4 dimers, which subse- (Fig. 4D). In addition, although the local overexpression of A20 quently activated NF-kB through MyD88 signaling (30, 31). Recent reduced brain edema and improved the NDS and neuron necrosis studies have indicated that A20 can disturb ubiquitin enzyme com- after ICH, the local overexpression of A20 may not be sufficient to plexes that are important for inhibiting NF-kB activation in TLR4 reduce the mortality after ICH. signaling. These studies indicated that the protective function of A20 8 ANTI-INFLAMMATORY ROLE OF A20 IN INTRACEREBRAL HEMORRHAGE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. A20 overexpression reduces inflammatory injury after ICH (A) Construction of the A20 overexpression mouse model. A20 expression in ipsilateral brain tissue following LV-A20 or LV-GFP injection was detected by (a1) qRT-PCR, (a2) Western blotting and (a3) immunofluorescence (green- EGFP, red-A20; scale bars, 100 mm). (B) Representative coronal sections from WT, LV-GFP, and LV-A20 mice (b1). The hematoma volume (b2) and hemoglobin content (b3) were calculated (*p , 0.05, **p , 0.01 versus WT.; n = 6 each). (C) Brain water content at sham and 3 d after ICH was compared between the WT, LV-GFP and LV-A20 groups (*p , 0.05, **p , 0.01 versus WT; n = 6 each). (D) NDS at 1, 3, 5, and 7 d after ICH was compared between the WT, LV-GFP and LV-A20 groups (**p , 0.01 versus WT; n = 20 each). (E) An ELISA Kit was used to determine IL-1b, IL-6 and TNF-a expression in WT, LV-GFP, and LV-A20 mice. (*p , 0.05, **p , 0.01 versus WT; n = 3 each). (F) There were no statistically significant differences in post-ICH mortality between the WT, LV-GFP, and LV-A20 groups (p . 0.05 versus WT, n = 20 each). (G) Representative TUNEL staining in WT, LV-GFP, and LV- A20 mice. Scale bars, 100 mm. The number of TUNEL-positive cells was calculated (**p , 0.01 versus WT, n = 3 each). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. A20 in peripheral blood may partially be involved in the regulation of ICH-induced inflammation. (A) A20 expression in PBMCs of A202/2 parabionts at 3 d after ICH. (B) Representative coronal sections from WT, WT parabiont, A202/2 parabiont and A202/2 mice (b1). The hematoma volume (b2) and hemoglobin content (b3) were calculated 3 d after ICH (*p , 0.05, **p , 0.01 versus WT, #p , 0.05, ##p , 0.01 versus A202/2, n = 6 each). (C) Brain water content at sham and 3 d after ICH was compared between the WT, WT parabiont, A202/2 parabiont and A202/2 groups. (*p , 0.05, **p , 0.01 versus WT, #p , 0.05 versus A202/2, n = 6 each). (D) An ELISA Kit was used to determine IL-1b, IL-6 and TNF-a expression at sham and 3 d after ICH in perihematomal tissue of the WT, WT parabiont, A202/2 parabiont and A202/2 groups. (*p , 0.05, **p , 0.01 versus WT, #p , 0.05 versus A202/2; n = 3 each). (E) Representative TUNEL staining in the indicated mice. Scale bars, 100 mm. The number of TUNEL-positive cells was calculated 3 d after ICH (#p , 0.05, **p , 0.01, n = 3 each). 10 ANTI-INFLAMMATORY ROLE OF A20 IN INTRACEREBRAL HEMORRHAGE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. A20 suppressed ICH-induced inflammatory injury by regulating the polyubiquitination of the E3 ubiquitin ligase TRAF6. (A) Interactions between TRAF6 and A20, Ubc13, UbcH5c, Itch, RNF11, and TAX1BP1 in the WT, A202/2, LV-A20 and LV-GFP groups at 1, 3, and 7 d after ICH. Proteins from lysates were immunoprecipitated with a TRAF6 Ab and detected by immunoblotting with Abs for A20, Ubc13, UbcH5c, Itch, RNF11, TAX1BP1, and TRAF6. (B) Lysates were subjected to immunoblotting with Abs to A20, Ubc13, UbcH5c, Itch, RNF11, TAX1BP1, IkBa, NF-kB p65 and GAPDH. (C) The specificity of the A20, Ubc13, UbcH5c, TAX1BP1, and TRAF6 interactions in an A20+/+ mouse for the indicated times. Proteins from lysates were immunoprecipitated with TRAF6 or a control rabbit Ab and detected by immunoblotting with Abs for A20, Ubc13, UbcH5c, Itch, RNF11, TAX1BP1, and TRAF6. may be mediated by the deubiquitination of A20 (14). In this report, embryonic fibroblasts, which had a very quick response; therefore, the authors performed in vitro experiments to confirm the molecu- stimulation was only performed for several hours. However, in our lar mechanisms of A20, and they used LPS to stimulate mouse study, we performed in vivo experiments to detect the molecular The Journal of Immunology 11 mechanisms of A20. Our and others’ previous work also showed that 6. Chen, S., Q. Yang, G. Chen, and J. H. Zhang. 2015. An update on inflammation in the acute phase of intracerebral hemorrhage. Transl. Stroke Res. 6: 4–8. the neuroinflammation was aseptic inflammation after ICH, and the 7. Guan, J., O. T. Miller, K. M. Waugh, D. C. McCarthy, P. D. Gluckman, and neuroinflammation changed over several hours to several days, A. J. Gunn. 2004. TGF beta-1 and neurological function after hypoxia-ischemia mostly reaching the highest levels at 3 d after ICH; therefore, in the in adult rats. Neuroreport 15: 961–964. 8. Liang, Q. 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Kanneganti, G. van Loo, and also differentially expressed in the indicated groups. Our results in- M. Lamkanfi. 2014. Negative regulation of the NLRP3 inflammasome by A20 dicate that A20 alleviated ICH-induced inflammation by inhibiting protects against arthritis. Nature 512: 69–73. 14. Shembade, N., A. Ma, and E. W. Harhaj. 2010. Inhibition of NF-kappaB signaling by Downloaded from TRAF6 polyubiquitination. A20 through disruption of ubiquitin enzyme complexes. Science 327: 1135–1139. In our study, we compared the inflammatory factors in the brain 15. Yang, Q. W., F. L. Lu, Y. Zhou, L. Wang, Q. Zhong, S. Lin, J. Xiang, J. C. Li, tissue of the WT, WT parabiont, A202/2 parabiont and A202/2 C. Q. Fang, and J. Z. Wang. 2011. HMBG1 mediates ischemia-reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. J. Cereb. Blood sham groups. 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Recommendations for the management of intracranial hae- 2/2 serious infection and high mortality, we did not perform A20 – morrhage - part I: spontaneous intracerebral haemorrhage. [Published erratum A202/2 parabiosis. appears in 2006 Cerebrovasc. Dis. 22: 461.] Cerebrovasc. Dis. 22: 294–316. 18. Lyden, P. D., A. Shuaib, K. R. Lees, A. Davalos, S. M. Davis, H. C. Diener, A20 can inhibit the activation of NF-kB, but the precise pathways J. C. Grotta, T. J. Ashwood, H. G. Hardemark, H. H. Svensson, et al; CHANT by which A20 induces NF-kB deactivation remain under investi- Trial Investigators. 2007. Safety and tolerability of NXY-059 for acute intrace- gation. TLR/IL-1R, IL-17R, and TCR signaling may participate in rebral hemorrhage: the CHANT trial. Stroke 38: 2262–2269. 19. Kamran, P., K.-I. Sereti, P. Zhao, S. R. Ali, I. L. Weissman, and R. Ardehali. this process (33–37). Our experiments mainly focused on TRAF6, 2013. Parabiosis in mice: a detailed protocol. J. Vis. Exp. 80: e50556. 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