Animal experiments
All the animal experiments were performed after obtaining approval from the Institutional Animal Ethics Committee of the National Brain Research Centre (NBRC) (NBRC/IAEC/2016/115 and NBRC/IAEC/2017/028). For in vivo experiments, postnatal day 8–10 (P08-P10) BALB/c mice were used, irrespective of their sex. The animals were handled in strict accordance with good animal practice as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forestry, Government of India.
IL-1β and morpholino treatment in mice
IL-1β was injected intraperitoneally (i.p.) at a dose of 10 ng/g of body weight of P10 BALB/c mice pups after every 24 h for different durations (1, 2, and 3 days) as described earlier [13]. Control mice group received intraperitoneal injection of equal volume of PBS.
Vivo-morpholino are morpholino oligos coupled with eight guanidinium head groups on dendrimer scaffold that enable delivery into cells [31]. Morpholino oligomers are proven antisense molecules used for the specific knockdown of the gene of interest both in vitro and in vivo. It either blocks the mRNA translation or interferes with RNA processing, including splicing and mRNA maturation [32]. HSP60 vivo-morpholino (HSP60-Mo) oligos were commercially procured from Gene Tools LLC (Philomath, OR, USA). HSP60-Mo was designed against sequences of mouse HSP60 (HSPD1) gene to specifically target it (5′ ACT GTG GGT AGT CGA TTT CT 3′). A 25-base scrambled morpholino of random sequence (SC-Mo) was used as a negative control (5′ TGG TTT CAG AAT AGT ATT CCA CTG C 3′).
For in vivo IL-1β experiments, animals were divided into six groups: (i) Control, (ii) IL-1β treatment, (iii) Sc-Mo, (iv) Sc-Mo + IL-1β treatment, (v) HSP60-Mo, and (vi) HSP60-Mo + IL-1β treatment group. Each group had a minimum of three animals. Among these, groups (v) and (vi) were intracranially injected with HSP60 vivo-morpholino at P8 (15 mg/kg of body weight of mice), while groups (iii) and (iv) received intracranial injection of scrambled vivo-morpholino at P8 (15 mg/kg of body weight of mice). As efficiency of vivo-morpholino in crossing the blood brain barrier is quite low, therefore, to achieve significant knockdown in the brain, our laboratory devised a slightly different strategy based on a previously published method [33, 34]. The intracranial injection was given manually in 8-day-old BALB/c mice pups (P8) at a single site as vivo-morpholino is believed to diffuse in the tissue [35, 36]. The amount of vivo-morpholino was calculated according to the body weight of each mice and calculated volume of vivo-morpholino was made up to 25 μl using 1× PBS. Then this 25 μl of vivo-morpholino solution was taken in the insulin syringe with needle size 31 G × 15/64 (0.25 × 6 mm), and it was slowly injected by gently pushing the syringe piston. Groups (i) and (ii) received an intracranial injection of PBS at P8 (same volume as vivo-morpholino). At P10, IL-1β was injected intraperitoneally (i.p.) in groups (ii), (iv), and (vi) at a dose of 10 ng/g of body weight of mice pups dissolved in 50 μl PBS, for three consecutive days. Groups (i), (iii), and (v) received same volume of PBS intraperitoneally. The mice were then sacrificed by repeated transcardial perfusion of chilled PBS and their brains were collected for protein and/or RNA analysis. The efficacy of the morpholino injection and its efficiency to knockdown HSP60 was checked through Western blot which was done by random sampling for morpholino-treated group. After we observed specific knockdown of HSP60 by vivo-morpholino (Additional file 1: Figure S1(A)), then only we proceeded for further experiments using vivo-morpholino with following four groups: (i) Control, (ii) IL-1β, (iii) HSP60-Mo, and (iv) HSP60-Mo + IL-1β groups.
Cell culture, IL-1β treatment, and transfections
All the in vitro experiments were performed in N9 murine microglial cells (N9 cells), which were a kind gift from Prof. Maria Pedroso de Lima (Center for Neuroscience and Cell Biology, University of Coimbra, Portugal) and were grown as described earlier [10]. N9 cells were chosen for the study as these microglial cells were derived from mouse brains and share many phenotypic characteristics with primary mouse microglia [37]. Transfection of HSP60 plasmid and endonuclease-prepared short interfering RNA (esiRNA) against mouse HSP60 gene was performed in N9 cells as described earlier for overexpression and knockdown experiments [10]. For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.
To induce inflammation, the N9 cells were serum starved for 2 h at 70% confluency and treated with 5 ng/ml IL-1β for different time periods. The cells were then used for different assays. For Western blotting, caspase-1 assay, and enzyme-linked immunosorbent assay, 1.5 × 106 cells were seeded in 90 mm × 20 mm plates, while for quantitative real-time PCR and flow cytometric analysis (reactive oxygen species analysis, cytokine bead array, and rhodamine 123 assays), 6 × 105 cells were seeded in 60 mm × 15 mm plates.
JEV infection of mice and N9 cells
Viral suspensions were prepared from the mice brain using the GP78 strain of JEV as described previously [38]. P10 BALB/c mouse pups were divided into six groups: (i) Control, (ii) JEV- infected, (iii) only Sc-Mo, (iv) Sc-Mo + JEV, (v) only HSP60-Mo, and (vi) HSP60-Mo + JEV group, and each group had a minimum of three pups. The HSP60-Mo group and HSP60-Mo + JEV infection group received an intracranial injection of HSP60-Mo at P8 (15 mg/kg of body weight of mice), while Sc-Mo and Sc-Mo + JEV groups were intracranially injected with scrambled vivo-morpholino (15 mg/kg of body weight of mice). Control and only JEV-infected groups received intracranial injection of PBS (same volume as vivo-morpholino) at P8. Mice from JEV group, Sc-Mo + JEV group, and HSP60-Mo + JEV group were injected with 1.5 × 103 plaque-forming units (PFU) of virus in 50 μl PBS, whereas control group, Sc-Mo group, and HSP60-Mo group were given same volume of PBS, intraperitoneally. After the development of full symptoms (including tremors, ruffled fur, hunching, ataxia, gait abnormalities like hind limb paralysis and body stiffening), the animals were sacrificed and their brains were excised after repeated transcardial perfusion with ice-cold PBS. The animal brains were then used for either protein or RNA analysis. Knockdown of HSP60 by vivo-morpholino was confirmed at protein levels by Western blotting (Additional file 1: Figure S1(B)). After confirming specific knockdown of HSP60 in JEV-infected group by HSP60 Mo, we proceeded with following four groups for further experiments: (i) Control, (ii) JEV-infected, (iii) only HSP60-Mo, and (iv) HSP60-Mo + JEV group.
For the JEV infection of N9 cells, around 1.5 × 106 cells were seeded in 90 mm × 20 mm plates in 5% RPMI and were allowed to grow for 12–15 h. After the cells reached 70% confluence, they were serum starved for 2 h and infected with JEV (strain GP78) at an MOI (multiplicity of infection) of 2 followed by incubation at 37 °C for 24 h to induce inflammation. The MOI of 2 was chosen for JEV infection as it significantly induces inflammation as compared to low MOI (Additional file 1: Figure S3). The cells were then harvested to isolate RNA for quantitative real-time PCR and protein for cytokine bead array and Western blotting.
Human brain tissues
Fresh frozen paraffin-embedded (FFPE) human brain tissue sections were obtained from the Human Brain Tissue Repository, National Institute of Mental Health and Neurosciences, Bangalore, India, in accordance with the institutional scientific ethics, protecting the confidentiality of the subjects. These sections were obtained from the frontal cortex/hippocampus postmortem from at least two confirmed patients of different brain disorders. For the control experimental sets, brain tissues from individuals who succumbed to traffic accidents and had no known prior neurological disease were used. The human glioma brain tissues were kindly provided by Dr. Ellora Sen (NBRC).
RNA isolation and quantitative real-time PCR (qRT-PCR) from tissues and cells
The 5-μm-thick FFPE frontal cortex sections were deparaffinized by repeated incubation in xylene followed by washing in alcohol gradient. Age-matched control samples were obtained from accidental cases with least possible trauma to brain. The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [10] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes. The conditions used for the qRT-PCR were as follows: 95 °C for 3 min (1 cycle) and 94 °C for 20 s, 55 °C for 30 s, and 72 °C for 45 s (40 cycles). The relative mRNA abundance was determined by normalizing to GAPDH mRNA using the Pfaffl method [39]. To elucidate the changes in IL-1β and HSP60 transcript levels in different brain disorders, two different qRT-PCRs were carried out (for IL-1β and HSP60) for each neurological condition. The qRT-PCR was done in triplicates. The primer sequences used for qRT-PCR analysis are listed in Additional file 1: Table S1.
Protein isolation
Cytosolic protein isolation
From N9 cells
Cytosolic protein fractions from N9 cells were isolated as described earlier [10, 13]. Briefly, around 3 × 106 were pelleted down and lysed in 100 μl lysis buffer containing 1% Triton-X-100, 10 mM Tris (hydroxymethyl) aminomethane-Cl (pH 8.0), 0.2% ethylene glycol tetraacetic acid, 1 mM ethylenediaminetetraacetic acid, 150 mM sodium chloride, 0.5% octylphenoxypolyethoxyethanol (Nonidet P-40), 0.2% sodium orthovanadate, and protease inhibitor cocktail (Sigma Aldrich). The samples were sonicated and the lysates were centrifuged at 12,000g for 30 min at 4 °C, followed by collection of supernatant containing cytosolic protein fraction. The protein was quantified using bicinconinic acid (BCA) method.
From BALB/c mice brains
For the cytosolic protein isolation from the brain samples, mice brain tissue was homogenized in 500 μl lysis buffer (composition mentioned above) to obtain cell suspension. The lysate was then sonicated and centrifuged at 12,000g for 30 min at 4 °C and supernatant was collected.
Nuclear protein isolation
From N9 cells
For the nuclear protein isolation, the untreated and treated cells were first lysed in buffer A (containing 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), nonionic surfactant, octylphenoxypolyethoxyethanol (IGEPAL), 0.2% sodium orthovanadate (SOV), and protease inhibitor cocktail (PIC) (Sigma Aldrich) for 30 min followed by centrifugation at 14,000g at 4 °C for 5 min. After discarding the supernatant, pellet was resuspended and sonicated in ice-cold buffer B containing 20 mM HEPES, 100 mM KCl, 1 mM EDTA, 0.2% SOV, and PIC. The lysate was centrifuged at 15,000g at 4 °C for 20 min. The nuclear extract was collected as supernatant and was estimated using BCA method.
From BALB/c mice brains
For the nuclear protein isolation from BALB/c mice brains, the whole brain tissues were first homogenized in buffer A (composition mentioned above) and the cell suspension was obtained. After this, the same protocol was followed to obtain nuclear protein from the brain cell suspension as used for in vitro N9 cell culture. The nuclear protein was then quantified by BCA method and was used for Western blotting.
Western blotting
Western blotting was performed as previously described [10]. Around 3 × 106 cells were pelleted and protein was isolated and quantified by the abovementioned protocol. For Western blotting of cytosolic as well as nuclear protein fractions, 30 μg protein was used. Primary antibodies against the following proteins were used: HSP60 (Abcam, #Ab46798), NLRP3 (Abcam, #Ab91525), inducible nitric oxide synthase (iNOS) (Abcam, #Ab3523), phospho-p65 nuclear factor-κB (NF-κB) p65 (S536) (Cell signaling Technology, #3033), Proliferating cell nuclear antigen (PCNA) (Cell Signaling Technology, #2586), Cycloxygenase-2 (COX2) (Millipore, #Ab5118), NF-κBp-65 (Santa Cruz Biotechnology, #SC372), and β-actin (Sigma Aldrich, #A3854). Secondary antibodies were labeled with horseradish peroxidase. The images were captured and analyzed using the Uvitec gel documentation system (Cambridge, UK) and ImageJ software respectively. Cytosolic protein levels were normalized to β-actin levels, whereas nuclear protein levels were normalized to PCNA. The fold change with respect to control cells was then calculated based on integrated density values (IDV).
Cytokine bead array (CBA)
For the quantitative analysis of various important cyto-chemokines in untreated and treated cells, the CBA kit (BD Biosciences, NJ, USA) was used. Around 1.5 × 106 cells were pelleted down and protein was isolated and quantified. The beads coated with antibodies against interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) antibodies were mixed with 50 μg cell lysates and standards according to the manufacturer’s instructions. The experiments were performed in triplicates as described earlier [13]. A total of 10,000 events were acquired for each sample. The results were analyzed using FACS Calibur (Becton Dickinson) and CBA software that allows the calculation of cytokine concentrations in unknown lysates with the help of a standard curve.
Reactive oxygen species (ROS) measurement
The levels of ROS generated within N9 cells of each treatment groups were measured by the CM-H2DCFDA (5 (and 6)-chlromethyl-20,70-dichloro-dihydrofluoresceindiacetate) (Sigma Aldrich), which is a cell-permeable, non-polar, H2O2-sensitive probe. It diffuses into cells, where intracellular esterases cleave its acetate groups, releasing the corresponding dichlorodihydrofluorescein derivative which gives red fluorescence [30]. 6 × 105 cells were seeded for the ROS analysis. After the completion of treatment, the untreated and treated N9 cells were incubated with 5 μM CM-H2DCFDA in dark at 37 °C room temperature for 20 min followed by washing and the relative mean fluorescence intensity was measured using FACS caliber (BD Biosciences, USA)). A total of 10,000 events were acquired in each treatment group.
Mitochondrial membrane depolarization assay
The integrity of the mitochondrial membrane was estimated by Rhodamine 123 (Rh 123) assay as described earlier [40]. Rh 123 is a cationic green fluorescent dye that can enter the mitochondrial matrix and the variation in the accumulation of Rh 123 in the cells is directly related to the change in the mitochondrial electrochemical potential (ΔψM). A decrease in the fluorescence of Rh 123 indicates a loss in the mitochondrial transmembrane potential and thus is a good method to identify mitochondrial damage. 6 × 105 cells were seeded for the Rh 123 assay. After the completion of treatment, control and treated N9 cells were incubated with Rh 123 (0.3 μg/ml) for 20 min at 37 °C, followed by washing and resuspension in FACS buffer. A total of 10,000 events were acquired in each treatment group on a flow cytometer (BD FACS Calibur, BD Biosciences, USA) and the relative mean fluorescence intensity of Rh 123 was assessed. Staurosporine (1 μM)-treated N9 cells were used as positive control (data not shown).
Caspase-1 activity assay
Levels of active caspase-1 were analyzed using caspase-1 activity assay kit (Millipore, USA, #21870) as per the manufacturer’s protocol. Briefly, around 3 × 106 cells were pelleted and resuspended for 10 min in 50 μl chilled lysis buffer followed by centrifugation at 10,000g at 4 °C for 1 min. The supernatant containing cell lysate was quantified using BCA method. Two hundred micrograms of the cell lysates were incubated with 50 μl of 2× reaction buffer and the substrate (YVAD-p-Nitroaniline, at a final concentration of 200 μM) at 37 °C for 2 h followed by measurement of absorbance at 405 nm to quantify caspase-1 activity levels in different treatment groups. This assay is based upon the spectrophotometric detection of the chromophore p-nitroaniline (p-NA) after cleavage from the YVAD-pNA substrate because of the activation of caspase-1.
Enzyme-linked immunosorbent assay (ELISA)
To quantify the levels of secreted IL-1β from different groups of N9 cells, ELISA was performed using mouse IL-1β ELISA kit (Biolegend, #432604) as per the manufacturer’s recommendations. Briefly, a rat monoclonal anti-mouse IL-1β capture antibody was coated in the 96-well plate overnight, followed by blocking for 1 h at room temperature (RT) and washing. For in vitro experiments, 1.5 × 106 cells were seeded in 90 mm × 20 mm culture plates and media was collected after the completion of treatments. For in vivo experiments, BALB/c brain lysates were used. Control and treated samples (100 μl media supernatant for in vitro and 100 μg protein from the mice brain lysates for in vivo experiments) were incubated in these wells overnight at 4 °C. The samples were then incubated with biotin conjugated detection antibody for 1 h at RT, followed by addition of avidin-HRP substrate for 30 min. The absorbance was measured at 450 nm on a spectrophotometer (Biorad, Australia), and the concentrations were calculated using the IL-1β standard reference curves.
Statistical analysis
Data are represented as the mean ± standard deviation (SD) from at least three independent experiments performed in triplicates (n = 3). The data was analyzed statistically by Student’s t test or one-way analysis of variance (ANOVA) followed by Holm-Sidak post hoc test. P value < 0.05 was considered significant. For in vivo treatments, a minimum of three mice were used in each group and experiments were repeated at least three times.
