SUPPRESSING NEUROINFLAMMATION USING NF-kB INHIBITORS USING BIO-TECHNE’S IMMUNOASSAY PLATFORMS TO INVESTIGATE NEUROINFLAMMATION APPLICATION NOTE In neurodegenerative diseases, such as Alzheimer’s and Parkin- son’s diseases, neuroinflammation has been shown to contribute A. to disease pathology.1,2 Microglia are the resident immune cells in the central nervous system (CNS) that initiate neuroinflamma- tion. As part of the innate immune system, microglia are respon- sible for surveying the local environment for danger signals.3 They express multiple pattern-recognition receptors (PRRs) that recognize endogenous damage-associated molecular patterns (DAMPs) and exogenous pathogen-associated molecular pat- terns (PAMPs). Stimulation of PRRs activate microglia, resulting in the secretion of proinflammatory cytokines, chemokines, and reactive oxygen and nitrogen species.2,4 One family of PRRs ex- pressed by microglia are the toll-like receptors (TLRs). Microglia express TLR1-9, and numerous studies have shown that these receptors, particularly TLR4, play a critical role in neuroinflamma- tion and neurodegenerative diseases.5-8 The TLR4 signaling pathway activates the NF-kB family of tran- scription factors. This protein family, which includes NF-kB1, NF-kB2, RelA/NF-kB p65, RelB and c-Rel, regulates several B. genes involved with inflammation including cytokines and che- mokines.9 Activation of NF-kB proteins require their release from inhibitory proteins that sequester the transcription factors in the cytoplasm. Two different groups of proteins bind and se- quester NF-kB proteins, the IkB family of inhibitors and the C- terminal portions of p105 and p100, which are the precursor proteins for NF-kB1 and NF-kB2, respectively.10 NF-kB proteins are activated by two distinct mechanisms. The canonical acti- vation of NF-kB proteins involves the inducible degradation of its IkB protein or IkB-like molecule p105 via phosphoryla- tion and subsequent Ubiquitin-mediated degradation. This al- lows the release and nuclear translocation of the canonical NF-kB family members, NF-kB1, RelA/NF-kB p65 and c-Rel.9-11 In contrast, noncanonical NF-kB signaling involves p100 process- ing. Activation of the noncanonical pathway results in the phos- Figure 1. The small molecule inhibitors Celastrol (A) and MLN4924 (Pevonedistat; B) phorylation and selective degradation of p100 by the Ubiquitin- were used to reduce LPS-induced inflammation in microglia. proteasome system, leading to the generation and subsequent nuclear translocation of NF-kB2.9-11 Interestingly, degradation of both IkBα, the most studied IkB protein, and p100 requires the activity of a SKP1-CUL1-F-box protein (SCF) E3 Ubiquitin li- gase.12,13 Additionally, SCF-mediated ubiquitination of IkBα and p100 is dependent on the neddylation of its CUL1 component by NAE (NEDD8-Activating Enzyme Inhibitor).12,14 In this study, we investigated if inhibition of the NF-kB signaling pathway could suppress neuroinflammation. Using mouse microglia BV-2 cells, we examined if MLN4924 (Pevonedistat), a protein neddylation inhibitor, and Celastrol, a known NF-kB inhibitor, could inhibit NF- kB-mediated production of cytokines and chemokines induced by LPS stimulation (Figure 1). MATERIALS AND METHODS CELL CULTURE AND TREATMENTS LUMINEX ANALYSIS Mouse microglia BV-2 cells were cultured in RPMI 1640 supple- Cell culture samples were analyzed for 26 different analytes us- mented with 10% FBS, 2mM L-glutamine, antibiotics (penicil- ing the Mouse Magnetic Luminex Assay (R&D Systems, Catalog lin 100 U/ml, streptomycin 100 µg/ml), and maintained in a 5% # LXSAMSM). Cell culture supernatants were diluted to 1:2 and CO2 incubator at 37 °C. Cells were left untreated or treated with 1:20 with the assay diluent, and cell culture lysates were run at 100 ng/mL LPS (Sigma, Catalog # L4516), 300 nM Celastrol (Tocris, 5 mg/mL per sample (125 µg/well). The assay was run according Catalog # 3203), 5 µM MLN4924 (R&D Systems, Catalog # I-502), to kit instructions and tested on a Luminex MAGPIX®. or DMSO for 24 hours. Cells were also pretreated with Celastrol (100 nM, 200 nM, 300 nM) or MLN4924 (1 µM, 5 µM, 10 µM) for SIMPLE WESTERN ANALYSIS 1 hour, washed with PBS, and then treated with 100 ng/mL LPS for 15 minutes or 24 hours. After each incubation period, condi- Whole cell lysates were loaded at 0.5 mg/ml and resolved un- tioned media and cells were separated by centrifugation. Condi- der reducing conditions using the 2-40 kDa separation sys- tioned media was aliquoted and stored at -20 °C until analysis. tem (ProteinSimple, Catalog # SM-W012). Capillaries were Cells were washed with PBS and then solubilized with Lysis Buffer probed using the following primary antibodies: Rabbit 17 (R&D Systems, Catalog # 895943) for Luminex® analysis, or Anti-Human Phospho-IkBα (S32/S36) Antigen Affinity-Purified Lysis Buffer 6 (R&D Systems, Catalog # 895561) for analysis using Polyclonal Antibody (R&D Systems, Catalog # AF4809) at the Simple Western™ platform. Lysis buffers were supplemented 50 mg/ml; Sheep Anti-Human/Mouse IkBα Antigen Affinity- with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Purified Polyclonal Antibody (R&D Systems, Catalog # AF4299) Scientific, Catalog # 78440) prior to lysis. Total protein of whole at 25 mg/ml and Rabbit Anti-Human/Mouse/Rat GAPDH Mono- cell lysates was determined via bicinchoninic acid assay (BCA). clonal Antibody (R&D Systems, Catalog # 2275-PC-100) at 5 mg/ml. Primary antibodies were detected using the Anti- CELL VIABILITY Rabbit Detection Module (ProteinSimple, Catalog # DM-001) or HRP-Conjugated Donkey Anti-Sheep IgG Secondary Antibody Cell viability was investigated for all experimental setups and aid- (R&D Systems, Catalog # HAF016) diluted 1:50 in Antibody Dilu- ed in determining optimal conditions. Viability was determined ent 2. Simple Western was visualized using chemiluminescence via trypan blue staining and assessed by the Countess™ II Auto- and evaluated using Compass Software. mated Cell Counter system. RESULTS AND DISCUSSIONS TLRs are thought to mediate the link between inflammation and abundant in the brain. They are activated by a broad range of neurodegenerative diseases. These highly conserved receptors stimuli and play key roles in a variety of diverse functions.11,18 are responsible for initiating and regulating innate immune re- Several CNS diseases and disorders have been linked to NF- sponses throughout the body including in the CNS. Activation kB activation.11,18,19 As a result, researchers are looking at the of TLRs results in the production of proinflammatory mediators NF-kB pathway as a potential therapeutic target for neurode- such as cytokines and chemokines.6 TLRs are typically activated generative diseases.20,21 In this study, we utilized our Luminex by specific PAMPs present in microbial molecules, or by DAMPS and Simple Western platforms to investigate if inhibition of the exposed on the surface of, or released by, damaged cells. How- NF-kB signaling pathway could suppress the neuroinflammatory ever, there is mounting evidence that TLRs expressed by neural response of LPS-stimulated mouse microglia BV-2 cells. cells, specifically TLR2 and TLR4 on microglia and astrocytes, can also be activated by aggregated proteins associated with neuro- Mouse microglia BV-2 cells were treated with 100 ng/mL of LPS for degenerative diseases including Amyloid b (Ab), α-Synuclein and 24 hours, and the levels of 26 different cytokines and chemo- mutant SOD1.8,15-17 Additionally, activation of TLRs by these ag- kines were measured in the cell culture supernatants using gregated proteins has been shown to potentiate disease patho- our Mouse Luminex Magnetic Assay (Figure 2). We observed genesis, in part, by the induction of chronic neuroinflammation.6 an increase in the levels of all analytes in the conditioned me- dia from LPS-treated cells (red bars) compared to untreated TLR signaling is complex. It involves the concerted action of an cells (blue bars). Pretreatment with Celastrol (300 nM; grey array of adaptor proteins, kinases and E3 Ubiquitin ligases, which bars) or MLN4924 (10 mM; orange bars) inhibited LPS-in- results in the activation of several different transcription factors duced secretion of several cytokines and chemokines by that regulate the expression of genes involved in the inflamma- mouse microglia BV-2 cells including IL-1α/IL-1F1, IL-1β/IL-1F2, tory response.7 One family of transcription factors that is activat- IL-6, TNF-α, CXCL10/IP-10/CRG-2, CCL4/MIP-1β, and CCL5/RAN- ed by TLR signaling is NF-kB. The NF-kB family of transcription TES. This decrease in cytokine expression was not due to a signifi- factors is composed of NF-kB1, NF-kB2, RelA/NF-kB p65, RelB cant change to total cells numbers (data not shown) or a decrease and c-Rel. These subunits form homo- or heterodimers that are in cell viability (Figure 3). retained in an inactive form in the cytoplasm. Activation of the canonical and non-canonical NF-kB signaling pathways results in release of the dimer and its subsequent translocation into the nucleus where it binds to DNA. NF-kB transcription factors are 35,000 350,000 30,000 300,000 25,000 250,000 200,000 20,000 150,000 15,000 100,000 10,000 50,000 (pg/mL) Concentration Analyte 5,000 Analyte Concentration Concentration (pg/mL) Analyte 0 0 CCL4/MIP-1β CCL2/JE/MCP-1 G-CSF IL-6 CXCL10/ CCL3/ CCL5/RANTES IP-10/CRG-2 MIP-1α 1,200 300 1,000 250 800 200 600 150 400 100 200 50 Analyte Concentration (pg/mL) Concentration Analyte Analyte Concentration (pg/mL) Concentration Analyte 0 0 IL-1α/IL-1F1 TNF-α CCL20/MIP-3α IL-27 CCL11/Eotaxin FGF basic/FGF2 IL-12p70 60 50 40 30 20 Analyte Concentration (pg/mL) Concentration Analyte 10 0 GM-CSF Granzyme B CXCL1/GROα/ IFN-γ IL-10 IL-13 IL-17/17A IL-3 IL-4 IL-5 CCL19/MIP-3β VEGF KC/CINC-1 Untreated LPS LPS + Celastrol LPS + MLN4924 Figure 2.