Both 8-week-old male and female hTNF-TG mice (Taconic Biosciences), male 10–12 week-old DBA/J1 mice (Jackson Laboratory), and male 8-week-old wild-type (WT) C57BL/6 mice (Jackson Laboratory) were used for experiments. Mice, 5 in a cage, were housed in a rodent barrier facility at Skirball Animal Facility of New York University Langone Medical Center on a 12-h light-dark cycle with ad libitum access to food and water. Mice with weight 18–22 g that were in good health condition were enrolled for experiments and no mice were excluded. When starting experiments, animals were randomly assigned to each group on the basis of random number table, six mice were assigned to each group based on empirical choice. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and use Committee of New York University School of Medicine.
Reagents and materials
Dulbecco’s modification of Eagle’s medium (DMEM, 10-017-CM) was purchased from Corning, and fetal bovine serum (FBS, S1620) was bought from Biowest. Human TNFα/TNFSF1A (NFTA0), IL-12 (419-ML-010), TGF-beta (240-B-002) were bought from R&D Systems. Penfluridol (PF, 26864-56-2) and methotrexate (59-05-2) were bought from MedChem Express. Type II chicken collagen (20012), complete Freund’s adjuvant (7001), and incomplete Freund’s adjuvant (7002) were purchased from Chondrex. Dextran sulphate sodium salt (DSS, molecular weight: 36000-50000 Da) was bought from MP Biomedicals. Picrylsulfonic acid solution (Synonym: 2,4,6-Trinitrobenzenesulfonic acid solution, TNBS, 92822), 5-aminosalicylic acid (5-ASA, A-3537), and LPS (L2630) were bought from Sigma-Aldrich. TransAM®NFkB p65 Activation Assay kit (40096) was bought from ACTIVE MOTIF. 3.5-French 38-cm catheter (4193505) was bought from Utah Medical Products, Inc. Plastic feeding tubes, 20 ga × 38 mm (FTP-20-38-50) were bought from INSTECH Laboratories. Antibodies against p-Erk1/2 (4370s), t-Erk1/2 (4695), p-p38 (9211s), t-p38 (9212), p-JNK (9255s), t-JNK (9258), t-p65 (4764s), and GAPDH (2118) were purchased from Cell Signaling Technology. Receptor activator of nuclear factor kappa-B ligand (RANKL, sc-9073) and Lamin B (sc-6216) were bought from Santa Cruz. RNeasy® Mini Kit (74106) and Random Hexamers (79236) were bought from Qiagen. TNFα (PHC3015), mouse IL-1β enzyme-linked immunosorbent assay (ELISA) kit (88-7013), and mouse IL-6 ELISA kit (88706476) were purchased from Invitrogen. VECTASHIELD® Mounting medium with DAPI (H-1200) was bought from Vector Laboratories. SYBR® Green PCR Master Mix (4309155) was bought from Applied Biosystems. ImProm-IITM Reverse Transcriptase (M314C) and Dual-Luciferase reporter assay system (E1910) were purchased from Promega Corporation. Nitrocellulose Membranes (0.45 μm, 162-0115) was bought from Bio-Rad. RIPA Lysis Buffer system (sc-24948A) was bought from ChemCruz. Macrophage Colony-Stimulating Factor (M-CSF, 576406), IFNγ (505702), IL-4 (504102), PE-CD25 (102008), anti-CD3 (100302), anti-CD28 (102102), anti-IL4 (504102), anti-IFNγ (505702), and recombinant mouse IL-6 (575702) were bought from Biolegend. ACK Lysing Buffer (A10492-01) was bought from Gibco. Pronase from Streptomyces griseus (PRON-RO) was bought from Roche. Acid Sphingomyelinase Assay Kit (K-3200) was bought from echelon. Antibody against acid SMase/SMPD1 (LS-C334919) was bought from LSBio. Silencer® Select Negative Control siRNA (4390843) was bought from life technologies. TRIzol reagent (15596026), mouse SMPD1 siRNA (151776, AM16708), and NE-PER nuclear and cytoplasmic extraction reagents (78833) were bought from Thermo Fisher Scientific. Fixation/Permeabilization Solution Kit with BD GolgiPlug™ (555028) was bought from BD Biosciences. Mouse Naïve CD4+T cell Isolation Kit (130-104-453) and MACS Separation Columns were bought from Miltenyi Biotec. FITC-CD4 (M1004502) was bought from Sungene. Percp-cy5.5-IFNγ (45-7311-82), PE-IL-17A (12-7177-81), and Alex-Fluo647-FoxP3 (51-5773-80) were bought from eBioscience. PE-IL4 (554389) was bought from BD Pharmingen.
BMDMs were treated with TNFα (10 ng/mL) in the presence or absence of PF (1μM) for 24 h. Cells were collected to extract total mRNA using RNeasy Plus Mini kit, and mRNA samples were sent to the Genome Technology Center at NYU Langone Medical Center for transcriptome sequencing. RNA-seq transcriptome library was prepared using 1 μg of total RNA. Messenger RNA was isolated based on polyA selection method and then fragmented by fragmentation buffer. Double-stranded cDNA was synthesized and was subjected to end-repair, phosphorylation, and “A” base addition. Libraries were size selected for cDNA target fragments of about 300 bp on 2% Low Range Ultra Agarose followed by PCR amplified for 15 PCR cycles. After quantification by TBS380, paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq xten sequencer (2 × 150 bp read length).
Those genes downregulated after TNFα stimulation and treatment with penfluridol were used to draw a heatmap and analyze the gene expression differences. We used the online tool TFactS (http://www.tfacts.org/) to identify the transcription factors that regulated those downregulated genes.
NF-κB luciferase assay
NF-κB luciferase assay was performed according to previously published methods . 293T cells were seeded in 24-well plate and transfected with NF-κB luciferase reporter gene plasmids (1.5 μg) and Renilla plasmids (0.3 μg) by lipofectamine2000 for 8 h. PF was added at concentrations of 0.001, 0.01, 0.1, 1.0, and 2.5μM overnight. The next day, cells were stimulated with TNFα (10 ng/mL) for 4 h. Measurements of luciferase activity were conducted based on the manufacturer’s specifications. Briefly, 100-μL luciferase assay reagents (LARII) were added to wells followed by 20-μL cell lysates and mixture with a pipette tip. Firefly luciferase was measured at a wavelength of 560 nm. After adding 100 μL Stop & Glo® Substrate, a second measurement for Renilla luciferase was performed at 480 nm. The fluorescence results were standardized to Renilla luciferase.
When inducing BMDMs, freshly isolated cells from the bone marrow were seeded in 6-well or 10-cm plates. BMDMs in 6-well plates were subjected to reduced serum conditions with DMEM supplemented with 2% FBS overnight, followed by adding DMSO or penfluridol (1 μM) for 2 h before stimulating with TNFα (10 ng/mL) for 15, 30, or 60 min. Protein samples were prepared by lysing cells in RIPA buffer containing PMSF, proteinase inhibitor cocktail, and NaVO4. After determining the protein concentrations by bicinchoninic acid (BCA) assay, the proteins were boiled for 5 min in SDS sample buffer. BMDMs in 10-well plates were maintained in DMEM with 2% FBS overnight, followed by adding DMSO or penfluridol (1 μM) for 2 h and then stimulated with TNFα (10 ng/mL) for 15, 30, or 60 min. Cytoplasmic extracts (CE) and nuclear extracts (NE) were prepared using a commercially available kit (NE-PER nuclear and cytoplasmic extraction reagents, 78833, Thermo Fisher Scientific). A total of 20 μg protein samples was separated by gel electrophoresis and transferred to a nitrocellulose membrane using a wet transfer system. The membrane was blocked in 5% (w/v) non-fat milk in TBST for half an hour at room temperature. Incubation with primary antibody was carried out overnight at 4 °C followed by three TBST washes and application of secondary antibody for 1 h at room temperature. The bands on the membrane were developed by enhanced chemiluminescent substrate and visualized by a gel scanner.
NF-κB DNA-binding activity
BMDMs were treated with PF (1 μM) overnight, then stimulated with TNFα (10 ng/mL) for 4 h. Cells were lysed and NF-κB DNA-binding activity was tested according to the manufacturer’s specifications (TransAM®NFkB p65 Activation Assay kit, 40096, ACTIVE MOTIF). Briefly, NF-κB transcription factor was captured by binding to a consensus sequence 5′-GGGACTTTCC-3′ which was immobilized on a 96-well plate. Nuclear extract (5 μg) was added to each well and incubated with anti-NF-κB p65 antibody, followed by HRP-conjugated secondary antibody incubation. The absorbance was read on a SpectraMax i3x plate reader at a wavelength of 450 nm. Results were expressed as the fold changes of NF-κB DNA-binding activity relative to control cells.
IL-1β and IL-6 levels in cell cultural supernatants or in sera from animal models were measured by mouse ELISA kits based on the manufacturer’s instructions. Optical density was measured by SpectraMax i3x plate reader at a wavelength of 450 nm, and concentrations were calculated according to the standard curve. Supernatants from cells were prepared by addition of TNFα (10 ng/mL) with or without PF (0.1μM or 1 μM) for 24 h.
Real-time quantitative PCR
Total RNA was extracted from inflamed joints and colons by TRIzol according to the manufacturer’s instructions. To prepare samples, 10-mg tissues were cut into small pieces and put into a grinding tube, then 1 mL TRIzol was added. Grinding of the prepared samples was performed with a tissue homogenizer, a PowerLyzer 24 (Qiagen). Samples were subsequently centrifuged at 10,000g for 5 min at room temperature, collecting the supernatant for mRNA extracting.
Total RNA in cells was extracted with RNeasy plus mini kit and cDNA was synthesized using SuperScript® Reverse Transcriptase. SYBR® Green PCR Master Mix was used to perform real-time quantitative PCR (qRT-PCR) on a StepOnePlusTM real-time PCR System (Applied Biosystems). The mRNA expression levels were calculated by ΔΔCT, and fold changes of target mRNA levels were normalized to GAPDH. The following specific sequences as SYBR primers were used for the target gene expansion: mouse IL-1β (5′–3′) F: AAT CTC ACA GCA GCA CAT CA, R: AAG GTG CTC ATG TCC TCA TC; mouse IL-6 (5′–3′) F: TTC CAT CCA GTT GCC TTC TTG, R: AGG TCT GTT GGG AGT GGT ATC; mouse NOS2 (5′–3′) F: TGT TAG AGA CAC TTC TGA GGC TC, R: ACT TTG GAT GGA TTT GAC TTT GAA G; mouse IL-17 (5′–3′) F: GGG AAG TTG GAC CAC CAC AT, R: TTC TCC ACC CGG AAA GTG AA; mouse MCP-1 (5′–3′) F: GTC CCT GTC ATG CTT CTG G, R: GCG TTA ACT GCA TCT GGC T; mouse CXCL10 (5′–3′) F: CCA AGT GCT GCC GTC ATT TTC, R: GGC TCG CAG GGA TGA TTT CAA; mouse GAPDH (5′–3′) F: AGA ACA TCA TCC CTG CAT CC, R: AGT TGC TGT TGA AGT CGC.
Isolation of bone marrow-derived macrophage
Bone marrow-derived macrophages (BMDMs) were prepared from WT C57BL/6 mice. Following sacrifice, femurs and tibias were collected and cleared of tissue using sterile gauze. Bones were cut to open the medullary cavity and then centrifuged at 13000g for 90 s to collect bone marrow cells. Cells were seeded in DMEM containing M-CSF (10 ng/mL), medium was refreshed every third day over a 6-day culture period. Then BMDMs were ready for experiments after stimulation with M-CSF for 6 days.
hTNFα transgenic (hTNF-TG) mouse model
Eight-week-old hTNF-TG mice, which express a human TNFα gene on a C57BL/6 background and can spontaneously develop arthritis, were used to test the treatment effect of penfluridol on a TNFα-dominant arthritis model [19,20,21]. Body weight as well as clinical and deformity scores were assessed weekly. Clinical scores were taken as the sums of the scores from digits, paws, wrists, and ankles, and the highest score for each mouse was 24. The detailed scoring systems were as follows: 0 = normal, 0.2 = swollen joint, for 20 digits; 0 = normal, 1 = noticeably swollen, 2 = severely swollen, for 4 paws; 0 = normal, 1 = noticeably swollen, 2 = severely swollen, for 2 wrists; 0 = normal, 2 = noticeably swollen, 4 = severely swollen with stiffness of ankle joint, for 2 ankles. Deformity was ranked on a 0 to 3 scale, corresponding to absence of deformity, mild deformity, moderate deformity, severe deformity, and ankylosis, with the total highest score equaling 12. Prior to administration of drugs, hTNF-TG mice were randomly classified into to three groups (n = 6/group): vehicle group, methotrexate group  (MTX, 2 mg/kg, a positive treatment control) and penfluridol group (10 mg/kg). MTX and penfluridol were dissolved in DMSO and stored at − 80 °C (for up to 3 months) after aliquoting. For in vivo application, drugs were diluted by a solution of water:ethanol:2% acetic acid at 8:3:1 (v/v), making the final volume of DMSO less than 5%. Drugs were delivered via oral gavage once a day using flexible plastic feeding tubes. For preventative treatment, drugs were given and clinical assessment began on the day when mice were 8 weeks old. For therapeutic treatment, drugs were given and clinical assessment began on the day when the average score was 8. At the end of treatment and observation, mice were sacrificed and sera, knees, and ankles were collected.
Collagen-induced arthritis model
DBA/1J male mice aged 10–12 weeks were used to establish collagen-induced arthritis (CIA) model [23, 24]. A 100 μL emulsification of type II chicken collagen and complete Freund’s adjuvant was injected intradermally by a 27-G syringe 1.5–2.0 cm away from the tail base. On the 21th day after the first immunization, a 100-μL booster of type II chicken collagen and incomplete Freund’s adjuvant were intradermally given. Clinical scores for erythema and swelling were recorded every other day thereafter until the end of the experiment. The scores were assessed according to the following standards: 0 = normal, 1 = mild swelling involving ankle, wrist, or one digit, 2 = mild swelling involving entire paw or more than two digits, 3 = moderate swelling from the ankle/wrist to entire foot/paw and all digits, 4 = severe swelling or ankylosing deformity of the whole ankle/wrist, foot/paw and digits. The total highest score for a mouse was 16, and a higher score indicated greater severity. Mice were randomly allocated into three groups: vehicle group, MTX (2 mg/kg), and penfluridol (10 mg/kg), with six mice in each group. For prevention groups, daily oral gavage of drugs was initiated on the 18th day after the first immunization while for therapeutic treatment groups, drug delivery began when clinical score had reached an average of 5 in each group. Mice were sacrificed at experimental endpoints, and sera and ankles were collected.
DSS-induced colitis model
Prior to initiation of dextran sulphate sodium (DSS)-induced colitis, 8-week-old C57BL/6 mice were randomly segregated into vehicle, 5-ASA (50 mg/kg, serving as a positive control), PF 0.4 mg/kg, PF 2 mg/kg, and PF10 mg/kg groups (6 mice in each group). Drugs were delivered via oral gavage once a day until sacrifice starting from 3 days before establishment of the DSS-induced colitis model via addition of 3% DSS drinking water for 5 days and followed by normal drinking water for 3 days [25, 26]. Scores of weight loss, stool consistency, and rectal bleeding were recorded every day beginning on the first day of 3% DSS water supplementation and continuing until sacrifice. Disease activity index was the sum of the scores on weight loss, stool consistency, and rectal bleeding, the higher score correlating to more serious disease activity . The detailed scoring systems were as follows: weight loss (0 = less than 5%, 1 = between 5 and 10%, 2 = between 10 and 15%, 3 = between 15 and 20%, 4 = over 20%), stool consistency (0 = normal, 2 = loose stool, 4 = diarrhea), rectal bleeding (0 = negative, 2 = blood trace, 4 = gross blood). At the end of the experiment, mice were sacrificed, and sera and colons were collected. Colon length was measured by a caliper.
TNBS-induced colitis model
To establish 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model , 8-week-old C57BL/6 mice were pre-sensitized by application of 150 μL 1% TNBS on the depilated back between the two forelimbs. Five days later, the mice were randomly assigned to five groups: vehicle, 5-ASA (50 mg/kg), penfluridol 0.4 mg/kg, penfluridol 2 mg/kg, and penfluridol 10 mg/kg, six mice in each group. One week after pre-sensitization, the TNBS-induced colitis model was established by intrarectal injection of 100 μL 3% TNBS. Drugs were supplied by oral gavage one time a day starting from the fifth day after pre-sensitization until the end of experiment. Body weight was recorded every day thereafter. Four days after modeling, mice were sacrificed and colons were collected.
H&E staining and quantitative analysis
Fresh colons were fixed in 10% formaldehyde for 24 h before processed for paraffin embedding. Sections (5μm) were cut from tissue blocks and went through deparaffinized, rehydrated, routine H&E staining, dehydrated, and cleared for mounting.
H&E-stained sections of ankle or knee joints were used to assess the severity of arthritis via scoring on the basis of inflammation, pannus formation, and cartilage damage . The inflammation scores were 0, 1, and 2 corresponding to normal, local inflammatory cell infiltration, obvious inflammatory cell infiltration that formed lymphoid aggregates, and edema, respectively. Scores for pannus formation and cartilage damage were as follows: 0 = normal, 1 = pannus formation without articular cartilage infiltration, 2 = pannus formation with articular cartilage infiltration.
H&E staining score of colons : the scores for colitis were the sum scores of inflammatory cell infiltration and intestinal wall structure integrity, the higher scores indicated more serious inflammation. For scores of inflammatory cell infiltration: 0 = normal, 1 = inflammatory cell only infiltrated to mucosa, 2 = inflammatory cell infiltrated to mucosa and sub-mucosa, 3 = inflammatory cell was found in the whole intestinal wall. Scores for intestinal wall structure integrity were assessed by the change of epithelial cell: 0 = normal, 1 = inflammatory cell was locally infiltrated, 2 = focally formed ulceration, 3 = extensively formed ulceration with or without granulation tissue or pseudo-polyps.
Prediction of penfluridol binding target
Two steps were needed to predict the binding target of penfluridol. Firstly, finding out the canonical SMILES of penfluridol by visiting the online website, https://www. ncbi.nlm.nih.gov/pccompound. Secondly, visiting the prediction website (http://www.swisstargetprediction.ch/) , an open website for drug target prediction, to find the possible binding target of penfluridol. The prediction result showed that the acid sphingomyelinase (ASM) was the target of penfluridol.
Drug affinity responsive target stability (DARTs) experiments were performed to determine the binding between penfluridol and ASM based on previous reports [31, 32]. Cells were lysed by lysis buffer (1× protease inhibitor cocktail, 50 mM sodium fluoride, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 69% M-PER, V: V) on ice for 10 min. Lysates were collected into a sterile pre-chilled 1.5-mL tube and centrifuged at 18,000g for 10 min at 4 °C, and the supernatant was transferred to a new 1.5-mL tube. After measuring the protein concentration by BCA assay, 99-μL cell lysates were mixed with 1 μL DMSO or 1 μL penfluridol (1 mM) and incubated with shaking for 30 min at room temperature. Cell lysate mixtures were then divided into 20-μL aliquots for addition of pronase at a concentration of 1:800 or 1:1600 (pronase: total protein). Digestion was carried out for 5 min at room temperature and stopped by addition of 20× protease inhibitor cocktail and incubation on ice for 10 min. Following digestion, samples were mixed with SDS-PAGE loading buffer and boiled for 5 min at 100 °C. The samples were used to perform western blotting and ASM target bands were visualized by enhanced chemiluminescent substrate using a gel scanner.
Acid sphingomyelinase activity
Assays for ASM activity were performed according to the manufacturer’s specifications. Briefly, Raw 264.7 cells at 70–80% confluence were treated with or without TNFα (10 ng/mL) overnight. After washing twice with phosphate-buffered saline, cells were collected into a 1.5-mL tube in cell collection buffer by cell scraper. Then cells were lysed by freeze-thaw in liquid nitrogen for three cycles, and cell lysates were centrifuged at 14,000 rpm at 4° for 10 min. Supernatants were collected, and protein concentration was detected by BCA assay and was adjusted to 1.5 mg/mL. In total, 20 μL of cell lysates was added to a 96-well plate in the presence of varied concentrations (0.1, 1, 10, 100 μM) of penfluridol and incubated on a shaker for 1 h at room temperature. The following steps were on the basis of the manufacturer’s specifications. Briefly, after incubating cell lysates with ASM substrate for 3 h at 37 °C and adding stop buffer, the plate was read at 360 ex/460 em. The activity of ASM in each group was calculated on the basis of standards.
Spleen naïve CD4+T cell differentiation
Induction of spleen naïve CD4+T cell differentiation into T cell subsets was performed according to previously reported methods [33, 34]. One day before cell differentiation induction, 1 mL PBS containing anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) was coated for TH1, TH2, and Treg and 1 mL PBS containing anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) was coated for TH17 in a 24-well plate and the plate was incubated at 4 °C overnight. The next day, spleens were isolated from WT C57BL/6 mice and spleen cells were isolated by grinding spleen on a 70-μm mesh and collected in PBS containing 1% FBS, then centrifuged at 1000 rmp for 5 min twice, followed by lysing red blood cells with ACK lysing buffer for 5 min on ice. After centrifugation, naïve CD4+T cells were isolated by CD4+T cell isolation kit in MACS separation columns according to the manufacturer’s specifications. Coating buffer was aspirated, and the plate was washed once with PBS prior to seeding CD4+T cells (1 × 106 per well) in 1.0 mL DMEM. The following cytokines were added to induce T cell differentiation: IL-2 (20 ng/mL), IL-12 (15 ng/mL), and anti-IL4 (5 μg/mL) for TH1; IL-2 (20 ng/mL), IL-4 (10 ng/mL), and anti-IFN γ (5 μg/mL) for TH2; IL-6 (20 ng/mL), TGFβ (3 ng/mL), anti-IFN γ (5 μg/mL), and anti-IL4 (5 μg/mL) for TH17; IL-2 (20 ng/mL), TGFβ (15 ng/mL), anti-IFN γ (5 μg/mL), and anti-IL4 (5 μg/mL) for Treg. At the same time, penfluridol (0.1μM) or penfluridol (1μM) were added. Four days later, cells were collected for flow cytometry analysis.
To test the differentiation, 1 μL Golgi stop buffer was added to each well for 4 h. T cell subset stain was based on the specfications of Fixation/Permeabilization Solution Kit. TH1 were stained with FITC-CD4 and Percp-cy5.5-IFNγ, TH2 were stained with FITC-CD4, PE-IL4, TH17 were stained with FITC-CD4 and PE-IL-17A, and Treg were stained with FITC-CD4, PE-CD25, and Alex-Fluo647-FoxP3. For intracellular staining, antibodies were diluted at 1:100 while, for cell surface staining, antibodies were diluted at 1:400. All samples were sent to NYU Langone Medical Center for detecting.
Assessment of clinical signs, colon length measurement, and histology scoring were performed by a pair of two investigators. Blinding was applied to one of the two investigators for the whole data collection process but not for data analysis. All data were organized and analyzed in GraphPad 8.0 and SPSS 22.0 statistics software. Data were expressed as mean with standard error of the mean. Statistical significance of the differences among groups was determined by one-way analysis of variance and Bonferroni post hoc test. The statistical differences of clinical scores among different groups were analyzed by repeated measures analysis of variance. p < 0.05 was considered as statistically significant.