Neutralizing peripheral circulating IL1β slows the progression of ALS in a lentivirus‐infected OPTNE478G mouse model

Abstract Background Amyotrophic lateral sclerosis (ALS) is irreversible and fatal within 3–5 years, with limited options for treatment. It is imperative to develop a symptom‐based treatment that may increase the survival of ALS patients and improve their quality of life. Inflammation status, especially elevated interleukin 1β (IL1β), has been reported to play a critical role in ALS progression. Our study determined that neutralizing circulating IL1β slows down the progression of ALS in an ALS mouse model. Methods The ALS mouse model was developed by microinjection of lentivirus‐carrying OPTNE478G (optineurin, a mutation from ALS patients) into the intra‐motor cortex of mice. Peripheral circulating IL1β was neutralized by injecting anti‐IL1β antibody into the tail vein. Enzyme‐linked immunosorbent assay (ELISA) and real‐time polymerase chain reaction (RT‐PCR) were carried out to determine the protein and gene expression levels of IL1β. TUNEL assay was used to assess the neural cell death. Immunofluorescent staining of MAP2 and CASP3 was accomplished to evaluate neuronal cell apoptosis. Glial fibrillary acidic protein staining was performed to analyze the number of astrocytes. Rotarod test, grip strength test, balance beam test, and footprint test were conducted to assess the locomotive function after anti‐IL1β treatment. Results The model revealed that neuroinflammation contributes to ALS progression. ALS mice exhibited elevated neuroinflammation and IL1β secretion. After anti‐IL1β treatment, ALS mice revealed decreased neural cell death and astrogliosis and gained improved muscle strength and motor ability. Conclusions Blocking IL1β is a promising strategy to slow down the progression of ALS.


| INTRODUC TI ON
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is fatal to patients within 3-5 years of onset. 1 Although the control of ALS progression has attracted much attention, there is no efficient curative strategy to deal with this devastating neurodegenerative disease. Riluzole and edaravone are the only drugs approved by the U.S. Food and Drug Administration for ALS treatment, which may prolong survival by several months. 2 Thus, it is imperative to develop new drugs and curative strategies to advance the motor behavior of ALS patients.
Inflammation has been found to play a critical role in ALS progression. 3 For instance, elevated interleukin 1β (IL1β) secretion was associated with ALS progression in a transgenic mouse model with SOD1 G93A (mutant superoxide dismutase 1). 4 A recent study demonstrated that polymorphisms of IL1β showed higher ALS susceptibility and high rate of disease progression. 5 In our previous study, we revealed that mice infected with lentivirus-carrying OPTN E478G , a mutation that was identified in ALS patients, had elevated neuroinflammation and increased IL1β secretion. This inflammation state resulted in neuronal cell death and impaired mobility of mice. 6 These studies suggest that blocking IL1β is beneficial for maintaining neuronal cell survival and improving ALS patients' quality of life.
IL1β, also known as leukocytic pyrogen, is a fever-producing cytokine and is an important mediator of the inflammatory response.
Here, we scrutinize the effects of blocking IL1β in our ALS mouse model. Our results reveal that blocking peripheral IL1β with the IL1β antibody significantly enhanced motor ability in the ALS mouse model. The outcomes suggest that blocking peripheral IL1β can ameliorate the impaired motor ability of ALS patients and improve their quality of life.

| Animalmanagement
Male mice weighing 18-25 g were purchased from Hunan SJA Laboratory Animal Co., Ltd. The mice were randomly allocated to three groups (n ≥ 3 per group): the CTRL + PBS (phosphate-buffered saline) group (control virus only), OPTN E478G + PBS group, and OPTN E478G + anti-IL1β group. Five mice per cage were housed at 22°C and 50% humidity under a 12-h light-dark cycle; 2.0 μl of lentivirus was injected into the intra-motor cortex using a small animal stereotaxic instrument (Yuyan, Shanghai, China). Ten days after surgery, anti-IL1β antibody (Bio X Cell BE0246, Lebanon, NH, USA) treatments were performed once a week and lasted for 1 month, which was reconstituted with PBS (200 μg/ml) followed by tail vein injection at a concentration of 1 μg/g bodyweight. Behavioral tests were carried out for 5 days. C.-G.H. monitored the group assignment at the different stages of the experiment (during assignment, experiment, outcome evaluation, and data analysis). Mice were euthanized after behavioral tests; 0.3% pentobarbital sodium (20 μl/g) was used to anesthetize mice. Then, heart perfusion was conducted with 30 ml of saline and 40 ml of 4% PFA (paraformaldehyde) in sequence. The brains were quickly obtained and postfixed for 4-6 h in 4% PFA. All animal experiments were performed according to the instructions of animal welfare designated by the World Organization for Animal Health.

| Intra-motorcortexdelivery
Male mice (C57BL/6) (age: 10 weeks; weight: 18-25 g) were used to develop the ALS mouse model by intra-motor cortex microinjection (Yuyan), as reported previously. 6 Briefly, we anesthetized the mice with 0.3% pentobarbital sodium (20 μl/g), stabilized them, and shaved the hair on the skull. Midline incision was executed. The syringe was inserted to reach the motor cortex region (2.0 mm anterior to the bregma, 1.5 mm farther from the midline, and 2.0 mm in depth); 2 μl of the virus was infused into the motor cortex (flow rate: 1.0 μl/3 min). Suture the skin and disinfection with iodophor.

| Tailveininjection
We used tail veil injection to deliver the anti-IL-1β antibody to the mice. The mice were stabilized on a mouse fixator. The tail vein was exposed to wipe with alcohol. The needle (with a hosepipe connected to a 1-ml syringe) was inserted into the tail vein at a small angle. It was confirmed that the needle was inserted into the vein by pumping back before infusion. The maximum liquid for tail vein injection was 100 μl per mouse.

| Packagingoflentivirus
Lentivirus production was carried out as described in previous studies. 6,7 293T cells were transfected with 10 μg of pCDH-OPTN E478G with auxiliary plasmids (∆8.91, VSVG) in a 10-cm plate. After 24-h transfection, the supernatants were collected, filtered, and concentrated (with one-fourth V pre-cold PEG8000 concentration buffer [146.1 g/L NaCl, 200 g/L PEG8000] for 4 h). After the pellets were centrifuged at 4000 rpm for 20 min, they were resuspended in 100 μl of PBS; 1 × 10 8 TU/ml of lentivirus was used in this study.

| Rotarodtest
Balance and motor coordination were assessed using rotarod test, as reported before. 8 The accelerating rate was set to 5.0 for 2 min on a DXP-3 accelerating rotarod (Chinese Academy of Medical Sciences).
The accelerating rate was kept at 50.0 rpm for further 2 min. The time was recorded before falling off from the apparatus. Three trials with a 20-min break were conducted for mice per day, and the trials were performed for 5 days.

| Balancebeamtest
Balance maintenance was investigated as reported before. 8,9 A balance beam (0.9 × 0.9 × 50 cm horizontal wooden bar) was positioned above the ground (40 cm) with a cage beneath. A dark box was kept at the terminal end encouraging the mouse to run toward it. The time taken to cross the beam was recorded, and the speed was calculated. Three trials with a 20-min break were conducted per day, and the trials were performed for 5 days.

| Footprinttest
Footprint test was performed as reported previously with minor modifications. 6,8 A tunnel (10.0 × 10.0 × 70.0 cm) was prepared with a white paper beneath. The mouse's paws were painted with red (forepaws) or black (hind paws) pigments. We recorded the length of the hindlimb stride, the length of the forelimb stride, the width of the front base, the width of the hind base, the speed to traverse the tunnel, and the overlap between the hindlimb and forelimb.

| Gripstrengthtest
Grip strength test was carried out as previously reported. 6,10 A steel wire (diameter: 9.0 mm, length: 50.0 mm) was hung above the ground (40.0 cm). A mouse was hung on the wire. The time that the mouse could hold on to the wire was recorded for a maximum of 5 min.
Three trials with a 20-min break were conducted per day, and the trials were performed for 5 days.

| Enzyme-linkedimmunosorbentassay
Mice brain tissue homogenates were harvested after the anti-IL1β antibody treatment. Enzyme-linked immunosorbent assay (ELISA) was performed as described in previous studies. 6,11 Briefly, we measured IL1β using the mouse IL1β Valukine ELISA kit (VAL601, R&D, USA). A Varioskan LUX multimode microplate reader (Thermo Fisher Scientific) was used to analyze chemiluminescence. Cytokine concentration (pg cytokine/mg total protein) was calculated by fitting the standard curve.

| TUNELassay
The TUNEL cell apoptosis detection kit (Beyotime C1091, China) was used to examine neuronal cell death, as reported before. 6 The paraffinized brain tissue slices were deparaffinized and rehydrated. They were digested with proteinase K (20.0 mg/ml) at room temperature for 20 min. Endogenous peroxidase was inactivated using H 2 O 2 (3% in PBS) at room temperature for 20 min. The slices were incubated with biotinylated dUTP in TdT enzyme buffer for 1 h at 37°C. Then, the reaction was stopped; the slices were washed with PBS, incubated with anti-digoxigenin peroxidase conjugate at room temperature for 30 min in a humid chamber, and subject to chromogenic reaction using diaminobenzidine. Finally, the slices were rinsed, stained with hematoxylin, dehydrated, and sealed for microscopic observation. 12 The positivestaining cells and the total number of cells were counted on Image-Pro; the average ratio of TUNEL+ cells to the proportion of the total number of cells in each visual field in the brain per section was calculated.

| Immunohistochemistry
Immunohistochemical examinations were carried out as reported before 6 ; 0.3% sodium pentobarbital (20 μl/g) was used to anesthetize the mice. The brains were obtained after heart perfusion and cryoprotected in 30% sucrose for examining the frozen sections. The samples were embedded in optimal cutting temperature and quick-

| Statisticalanalysis
GraphPad Prism 7 was used for data analysis. Data are presented as mean ± standard deviation. Distribution style was checked using Shapiro-Wilk normality test. Multiple comparisons were carried out using analysis of variance (ANOVA), with one-way ANOVA test employed when only one variance was present and two-way ANOVA employed when more than one variance was present. Dunnett's multiple post-hoc comparison tests were performed to address statistical differences. Results were presented based on the median value and its quartiles in the whisker boxplots. Statistical significance was considered when p-value ≤0.05. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001; "ns" means no significance.

| IL1β was neutralized in the brain of lentivirusinfected OPTN E478G mice after anti-IL1β treatment
Preventing the progress of ALS remains a challenge. IL1β, an inflammatory factor, is elevated in disease conditions. The increased production of IL1β has been found in ALS, 4,6 and IL1β has been investigated as a potential therapeutic target for the treatment of ALS. 13 However, the effects the of anti-IL1β antibody treatment for ALS have not been investigated. To determine the therapeutic effects of the anti-IL1β treatment on ALS, we treated a previously established ALS mouse model with an anti-IL1β antibody. The mouse model was developed by microinjecting lentivirus-carrying OPTN E478G into the intra-motor cortex of mice. 6 OPTN E478G was found to be mutated in ALS patients and led to the elevated IL1β level in the mouse model. 6,14 Anti-IL1β (Bio X Cell BE0246) was reconstituted with PBS (200 μg/ml) and then injected into the tail vein of mice (1 μg/g bodyweight) to neutralize the peripheral IL1β after 10 days of lentivirus microinjection. Anti-IL1β antibody treatments were performed once a week four times. Behavior tests were conducted 7 days after the last anti-IL1β treatment to evaluate the effects of neutralization of IL1β on the ALS mouse model, and the samples were harvested after these tests. The time schedule is shown in Figure 1A. The intramotor cortex injection site is shown in Figure 2B. We found that IL1β was neutralized to the basal level after the anti-IL1β treatment ( Figure 1C). The mRNA expression of IL1β was also determined using qrt-PCR, showing that there was no difference with or without anti-IL1β treatment in the ALS mouse model. The results suggest that the anti-IL1β antibody efficiently downregulated IL1β in brain tissue in the ALS mouse model.

F I G U R E 1
Anti-IL1β treatment can reduce IL1β (interleukin 1β) in mouse brain tissue. (A) Diagram of the treatment of mice for subsequent analysis. Anti-IL1β infusion started after 10 days of lentivirus microinjection. Anti-IL1β treatment was performed once a week and lasted for 1 month. Assessments of movements started 7 days after last anti-IL1β treatment. The assessments were performed for five consecutive days. (B) The intra-motor cortex injection site is present by referring the mouse brain in stereotaxic coordinates (2 mm anterior to the bregma, 1.5 mm beside the midline, 2 mm in depth from the pia). 20 (C) IL1β in the brain tissue was analyzed using ELISA (enzyme-linked immunosorbent assay, left) and qPCR (quantitative polymerase chain reaction, right) after anti-IL1β treatment. Values are expressed as mean ± standard deviation, n = 3. Data analysis was performed by comparing CTRL and OPTN E478G + IL1β to the OPTN E478G group using oneway ANOVA (analysis of variance) with Dunnett's post-hoc test. ***p < 0.001; the absence of asterisks means no significance p > 0.05. To investigate the effects of anti-IL1β treatment on neural cells, we evaluated neural cell death and astrogliosis, which were the pathogenic features of the OPTN E478G mouse model. After the anti-IL1β treatment, OPTN E478G mice exhibited decreased neuronal cell death, as evidenced by TUNEL assay (Figure 2A,B). Immunofluorescent staining of MAP2 and CASP3, which were markers of neuronal cell and apoptosis, respectively, revealed that the ratio of CASP3 + to MAP2 + cells decreased by 50% when OPTN E478G mice were treated with the anti-IL1β antibody ( Figure 2C,D). Immunofluorescent staining of GFAP (an astrocyte marker) was substantially decreased, indicating alleviated astrogliosis around the degenerating motor neurons after the anti-IL1β treatment ( Figure 2E,F). These results suggest that neutralizing peripheral IL1β with an anti-IL1β antibody can ameliorate the inflammatory effects on neuronal cell death in the OPTN E478G mouse model.

| Anti-IL1β treatment improved the motor abilityofOPTN E478G mice
To investigate whether OPTN E478G mice could benefit from the anti-IL1β treatment, we performed an accelerating-rotarod test and a balance beam test to evaluate motor coordination and balance, a grip strength test to determine muscle tension, and a footprint test to evaluate mouse gait. After the anti-IL1β treatment, the latency to fall increased significantly, as measured by the accelerating-rotarod test ( Figure 3A); muscle tension also improved, as evidenced by longer latency to fall in the grip strength test ( Figure 3B); the speed of crossing the beam was faster in the balance beam test ( Figure 3C  ing IL1β with an anti-IL1β antibody was effective in slowing down ALS progression. This effect is expected to improve by using the intra-motor cortex microinjection approach. Another explanation is that perhaps the inflammation regulation in the peripheral system indirectly affects the central nerve system. Increased IL1β in brain tissue may (1) disrupt the permeability and integrity of blood-brain barrier 18 or (2) be transported to the peripheral system through the cytokine transporter. 18 Anti-IL1β treatment (intravenous) may exert its function on the central nervous system by (1) transducing the decreased IL1β signaling to the hypothalamus through vagal afferents or (2) preventing production of second messengers such as nitric oxide and prostaglandin. 19 Overall, our study suggests that blocking

| DISCUSS ION
IL1β is a promising strategy to slow down the progression of ALS.

| CON CLUS IONS
This study suggests that the inhibition of inflammation with an anti-IL1β antibody can benefit neuronal cell survival and ameliorate ALS progression in mice infected with lentivirus-carrying OPTN E478G .
Thus, curing ALS by targeting IL1β is a promising treatment.

AUTH O RCO NTR I B UTI O N S
Zheng-Zhao Liu and Wen-Bao Hu conceived the study, designed the experimental procedures, analyzed the data, prepared the manuscript, and supervised the project. Xin Wang, Zhi-Lin Pang, and Chun-Gu Hong performed the experiments and analyzed data. Ran Duan prepared the manuscript.

ACK N OWLED G M ENTS
We thank Rong-Gui Hu at the University of Chinese Academy of Science for giving valuable suggestions and providing technical support. This work was supported by grants from the National Natural Science Foundation of China (grant numbers: 82172502 and 81974127).

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

E TH I C S S TATEM ENT
The Ethical Review Board at Xiangya Hospital of Central South University approved the animal experiments (no. 201803512).