Chemicals and kits
Cell culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Protein assay kit was purchased from Bio-Rad (Hercules, CA, U.S.A.). TNF-α, IL-10, and IL-1β immunoassay kits were purchased from eBioscience (San Diego, CA, U.S.A.). The kits were used according to manufacturer´s instructions. Routine reagents and LPS from Escherichia coli (O111:B4, L2630) were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.) and recombinant α-Klotho from R&D Systems (1819-KL-050, Minnesota, MN, U.S.A.). All solutions were prepared immediately before use.
Primary cell culture
Primary mixed cortical glia culture was prepared as previously described . Brifely, cortex from newborn C57BL/6J mice (postnatal days 1–4) was dissected in ice-cold Hanks’ balanced salt solution (HBSS) under a microscope and their meninges removed. Small pieces of cortices were incubated in a trypsin solution (GIBCO) for dissociation at 37°C for 20 minutes. DMEM (complemented with glutamine, 10% Hyclone FetalLOne III serum, GE Healthcare and 1% Penicillin/ Streptomycin) was added to the solution to inhibit trypsin action, and cells were dissociated with a Pasteur pipette. The cells were passed through Cell Strainer, and cells were counted in a Neubauer chamber, and each Flask T75 (Sarstedt) was plated with 1x106 cells. The medium was changed every three days and kept for 10–14 days. The culture was plated in a 6-well plate or 24-well plates for experiments. The culture used for the experiments was considered an astroglial-enriched culture based on previous data showing 91,2% astrocytes labeled with GFAP . To obtain astrocyte-pure cell cultures (> 98%) for NF-kB experiments, flasks were incubated in an orbital shaker at 37°C, 180 r/min, for 15 h as previously described .
Primary cortical neuronal culture was prepared from both male and female postnatal (P1-3) mice C57BL/6 (Mus musculus). The meninges were separated from the brain, and the cortex were cut into small pieces and incubated in trypsin solution (2 mg/mL) for 20 min in a 37 ͦC, 5% CO2 incubator. After removal of trypsin solution, tissue was washed twicce with HBSS.Tissue was dissociated in HBSS containing 0.1 mg/mLDNAse by mechanical trituration with glass pipette. Cells were counted and plate (1x106 cells) in polyethylene (Sigma-Aldrich) pre-coated dishes. Neuons were maintained for wwo weks in Neurobasal medium (GIBCO) supplement with B27 (GIBCO), 2mM gluatamine, 100 U/mL penicillin,100 mg/mL streptomycin and 0.25mg/mL amphotericin B
Both cell culture preparation (glial cells and neurons) was conducted in accordance with The Ethical Principle in Animal Research adopted by the Brazilian College of Animal Experimentation (CONCEA) and were approved by the Ethical Committee for Animal Research (CEEA) of the Biomedical Sciences Institute of the Universidade de São Paulo, São Paulo, São Paulo State, Brazil. The protocol was registered under number 12/2016 CEEA of animals used for experimentation.
α-Klotho and LPS treatment
On the 15th day, the cells were treated with only DMEM without fetal bovine serum (FBS), LPS, α-Klotho (AA 35–982), or LPS + α-Klotho for 24 hours. A dose-response of recombinant α-Klotho and LPS treatments were made with different concentrations ( α-Klotho 0.1 nM – 4.0 nM) and LPS (0,01µg/mL-100µg/mL) for 24 hours to observe cell viability. After this first screening, only α-Klotho concentrations (0.1–2.0 nM) were tested at different time points (1, 4, and 24 hours) to evaluate the α-Klotho effect in LPS (1µg/mL /8 hours) -induced changes in TNF-α levels. Based on these data, the concentration of 1nM of α-Klotho and 1µg/mL to LPS for 4 and 8 hours was used to evaluate changes in NF-kB activity and IL-1β, IL-6 and IFN-γ levels, respectively.
For the experiments involving Glial conditioned Medium (GCM), on the 15th day, cell culture was pretreated with DMEM without FBS in the absence (control) or in the presence of α-Klotho (1 nM) for 24 hours, and then challenged with 1µg/mL LPS for 8 hours. The media were collected and named GCM or GCM-KL. Primary neuronal culture cells were challenged on the 10th day, by changing normal conditioned medium by 25% or 50% of GCM or GCM-KL for 24 hours.
Cell viability by LDH and MTT
Cell viability was estimated by Cytotox 96 non-radioactive assay (Promega). The assay was performed according to the Manufacturer's instructions. Lactate dehydrogenase (LDH) release was assayed after 24 hours of treatment with α-Klotho or LPS by removing 50µl supernatant from each well into a 96-well plate and incubating with cytotox 96 reagents for 30 minutes covered with aluminum foil at room temperature. The stop solution was added, and the absorbance was measured at 490 nm. The percentage of LDH activity was measured by the ratio: (Absorbance of the sample/ Absorbance of maximum activity) x 100%.
The cells were incubated with filtered MTT in DMEM without FBS at 37°C for 2 hours and 30 minutes. The supernatant was removed from the plate, and DMSO was added. The new supernatant was plated on a 96-well plate and measured at 570nm. MTT % related to control was calculated by the ratio: (absorbance of the sample - absorbance of DMSO)/ (absorbance of the control- absorbance of DMSO) x100%.
Multiplex analysis of cytokines and chemokines
Concentrations of TNF-α, IL-1β, IL-6, IL-18, and INF-γ were simultaneously measured in 25 µL of medium from homogenized cells using a Milliplex M.A.P. kit Mice Cytokine/Chemokine Magnetic Bead Panel (Millipore, Billerica, MA) by following manufacturer’s instructions. Antibody immobilized beads were detected on a Luminex 100 xMAP technology machine (Austin, TX). Standard curves were generated for each cytokine/chemokine using standards included in the kit for serum samples. The median fluorescence intensity for each analyte was calculated using a five-point logistic parameter curve, and normalized to the amount of protein in each sample.
For evaluate the effects of α-Klotho on NF-κB activation by LPS, after purification, astrocytes were treated for 24 hours with vehicle (control) (PBS) or 1 nM α-Klotho. Inflammatory stimulation with LPS 1 µg/ml was then performed and the cells fixed with methanol (10 min) 4 hours later and washed with PBS three times for 5 min. The fixed cells were incubated with serum blocking (5% normal donkey serum in triton X-100 0.01%) for one hour and incubated overnight with primary antibodies GFAP (1:300) (3670; Cell Signaling, and RelA (p65 (1:100) (ab7970; ABCAM, USA). The primary antibody was removed, and the plate was washed with serum blocking three times for 10 minutes. The cells were incubated with secondary antibody (rabbit or mouse anti donkey- Alexa 594 or 488, Thermo Fischer Scientific,1:1000), diluted in PBS with Triton X-100 0.01% for 2 hours, protected from the light. The coverslips were washed five times with PBS for 5 min and incubated with DAPI (4'-6-Diamidino-2- phenylindole; Sigma) for 1 minute in dilution 1:10.000. Slices were transferred to glass slides and analyzed in Fluorescence microscope Nikon Eclipse 80i (Nikon, Tokyo, Japan) with a camera system, Nikon Digital Camera DXM 1200C.
Protein extraction - nucleus and cytoplasm
Culture media were removed from 6-well plate, and the cells were scraped in cold PBS with 0.5mM PMSF and centrifuged at 4°C for 2 min at 13,000g. Pellet was resuspended in lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM PMSF, 0.1 mM EDTA, 2 µg/mL leupeptin, 2 µg/mL antipain, 30 mM NaF, 3 mM sodium orthovanadate, 20 mM sodium pyrophosphate and 5 mM BG-P) and incubated on ice for 15 min. NP-40 was added, and the samples were homogenized and centrifuged for the 30s at 13,000g at 4°C. Supernatants were used for Western blotting assay and pellets were resuspended in extraction buffer (1.5 mM MgCl2, 20 mM HEPES, pH 7.9, 25% glycerol, 300 mM NaCl, 0.5 mM PMSF, 0.25 mM EDTA, 2 µg/mL leupeptin, 2 µg/mL antipain, 3 mM sodium orthovanadate, 30 mM NaF, 20 mM sodium pyrophosphate and 5mM BG-P) and kept on ice for 20 minutes. Samples were centrifuged for 20 min at 13,000g at 4°C and supernatants were aliquoted as nuclear extract used for EMSA assay. Protein concentration was determined using the Bradford protein reagent (BioRad) .
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from control or treated cells were prepared as previously described . Doublestranded oligonucleotide containing the NF-κB consensus sequence from Promega (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was end labelled using T4 polynucleotide kinase (Promega) in the presence of γ-32P dATP. Nuclear extracts (2.5 µg) were incubated with 32P-labelled NF-κB probe. The binding reaction was performed at room temperature for 30 min in a reaction buffer containing 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM MgCl2, 2.5 mM EDTA, 20% glycerol, 0.25 µg/µL of poly (dI-dC) and 2.5 mM dithiothreitol. DNA protein complexes were separated by electrophoresis through a 6% acrylamide:bis-acrylamide (37.5:1) gel in TBE (45 mM Tris, 45 mM boric acid, 0.5 mM EDTA) for 2 h at 150 V. Gels were vacuum dried for 1 h at 80°C and exposed to X-ray film at − 80°C. For competition assays, nuclear extract was incubated with specific competitor (unlabelled double-stranded NF-κB consensus oligonucleotide) or a non-specific competitor (unlabelled transcription initiation factor IID [TFIID]). For supershift assay, antibodies against subunits of NF-κB (p50 and p65,1:20) (Santa Cruz Biotechnology) were added into the binding reactions. Autoradiographs were visualized using a photodocumentation system DP-001-FDC and quantified with ImageJ (NIH) software 70.
Electrophoresis was performed using 10% polyacrylamide gel and the Bio-Rad mini-Protean III apparatus (Bio-Rad, Hercules, CA, U.S.A.). In brief, the proteins present in cytosolic and nuclear fractions were size-separated in 10% SDS-PAGE (90 V). The immunoblotting was performed as described previously The proteins were blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, U.S.A.) and incubated with the specific antibody: RelA (p65) (1:1000, sc-0372; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (1:2000 (58169; Cell Signaling, U.S.A.) and after with secondary antibody (Rabbit). Proteins recognized by antibodies were revealed by an electrochemiluminescence (ECL) technique, following the Manufacturer's instructions (Amersham Biosciences, Amersham, U.K.). To standardize and quantify the immunoblots, we used the photo documentation system DP-001-FDC (VilberLourmat, Torcy, France) and N.I.H. ImageJ software (http://rsb.info.nih.gov/ij). Several exposure times were analyzed to ensure the linearity of the band intensities.
Results are expressed as mean ± S.E.M. of the indicated number of experiments. Statistical comparisons for α-Klotho-induced changes in cytokines, Western blotting, and cell viability were performed by one-way analysis of variance (ANOVA), followed by the Tukey post-test. All analyses were performed using a Prism 9 software package (GraphPad Software, San Diego, CA, U.S.A.). P-values < 0.05. were considered to reflect a statistically significant difference.