Selective ROCK2 inhibitors: KD025 (Selleck Chemicals LLC, #S7936) and GV101 (Beijing Tide Pharmaceuticals) and pan-ROCK inhibitors: H-1152 (Tocris Bioscience) and GSK429286 (MedChemExpress) were dissolved in DMSO and stored at −20 °C until used in cell-based assays. In addition to ROCK1, GV101 at 10 μM concentration was found to have no more than 50% inhibition of 50 intracellular kinases: MuSK(h), DRAK1(h), ALK2(h), LKB1(h), PDK1(h), TrkA(h), Fyn(h), CLK1(h), MSK1(h),PASK(h), Aurora-A(h), Flt3(h), p70S6K(h), TYK2(h), LIMK1(h), SAPK2a(h), activated IGF-1R(h), MLCK(h), Rsk1(h), AMPKα2(h), DYRK1B(h), GSK3α(h), JAK1(h), MRCKα(h), KDR(h), PKBα(h), Haspin(h), CK2α1(h), PDGFRα(h), DDR1(h), mTOR/FKBP12(h), PI3 Kinase (p110a/p85a)(h), CK2α2(h), LOK(h), MSSK1(h), IR(h), AMPKα1(h), GRK2(h), PKCα(h), JAK3(h), MRCKβ(h), PKCμ(h), Blk(h), PKG1β(h), Ron(h), Itk(h), PKA(h), ZAP-70(h), PKBγ(h), PKCθ(h), DMPK(h), Rsk2(h), Syk(h), PRK2(h), Fms(h), PKAcβ(h), Lck(h).
Female C57BL/6 mice six to seven weeks of age were obtained from Charles River. Animals were housed in a temperature-controlled room with a light/dark cycle of 12 h and with ad libitum access to food and water throughout the study. On the day of the study start, the animals were divided into two main groups, with one group (n = 15) receiving a normal drinking water and normal chow and the other group (n = 75) receiving TAA (Sigma Cat# 163678) supplemented water alone for 9 weeks or in combination with Western Diet (WD) chow for 12 weeks (21.1% fat, 41% Sucrose, and 1.25% Cholesterol by weight) purchased from Teklad diets (TD; #12052). At the beginning of week 7, 55 animals provided with TAA- supplemented water alone or in combination with WD were distributed into 5 treatment groups of ten animals each. Five animals from normal drinking water/chow group and TAA-water/TAA with WD group were harvested on week 6 before treatment initiation. On week 7 post randomization, animals were administered with indicated doses of GV101, KD025 or vehicle (0.5%CMC-Na, 800–1200) once daily via oral route for three weeks (week 7–week 9) or six weeks (week 7–week 12). Animals were harvested on week 9 after provided TAA supplemented water or week 12 after provided TAA supplemented water and WD. At end point, serum and tissues were collected, snap frozen or fixed, and fibrosis-related symptoms were analyzed. Individual animals were monitored daily for clinical observations, including general activity levels and morbidity. Any signs of discomfort were documented. Body weight was recorded twice weekly throughout the study period. This study was performed at Aragen Bioscience under the Animal Use Protocol: AUP#: 18-0803-MR. We have complied with all relevant ethical regulations for animal testing.
Tissue and serum analysis
Hydroxyproline analysis was performed by using median liver lobes snap frozen at harvest. The Western blot assessment was performed by using total spleen and caudate liver lobe tissues. The left liver lobes were fixed in 10% neutral buffered formalin and paraffin-embedded blocks were processed for histological analysis. For each tissue, 2 series of 2 sections (5 µm thick) were cut longitudinally and spaced by 50 µm. The two sections were laid on a same slide, stained with picrosirius red (PSR). Slides were scanned using the NanoZoomer-SQ scanner (Hamamatsu), at the magnification of X20 (0.452 µm/pixel) and digital slides of whole sections captured using NDP.view 2 Hamamatsu software. The drug levels were measured in the right liver lobes and serum. Cytokine and liver enzyme analysis was performed by using serum samples.
Isolation and stimulation of human primary immune cells: PBMCs, T cells, monocytes and monocytes-derived macrophages
All human primary immune cells were purified from the peripheral blood (Leukopak) of healthy human donors between ages of 16 and 75 years (New York Blood Center, NY) by centrifugation at 1200 g for 20 min without brake over a Ficoll cushion. Human T cells were purified using RosetteSep™ Human CD4+ T cell Enrichment Cocktail (StemCell Technologies #15062), Vancouver, BC, Canada) before centrifugation, as previously described10. Human primary monocytes were isolated using RosetteSep Human Monocytes Enrichment Cocktail (StemCell Technologies #15068) before centrifugation. PBMCs were isolated directly by similar centrifugation. Purified PBMCs, T cells or monocytes were washed and centrifuged 2 times at 300 g and one last time at 100 g for 10 min without brake to help remove platelets. Immune cells were finally resuspended in RPMI containing 10% FBS and 1% Penicillin/Streptomycin (the immune cells medium) and counted.
For Th17-skewing activation, freshly purified CD4+ T cells were cultured in immune cells medium at a final concentration of 2 × 106/ml and stimulated with anti-CD3 mAb (5 μg/ml; eBioscience, San Diego, CA) and anti-CD28 mAb (5 μg/ml; eBioscience, San Diego, CA) in combination with IL-1β (50 ng/ml; R&D Systems Inc, Minneapolis, MN) and TGF-β (5 ng/ml; R&D Systems Inc, Minneapolis, MN) with or without indicated doses of ROCK inhibitors. Cytokine secretion was determined by ELISA after 48 h by using IL-17, IL-21 and CXCL13 kits. The percentage of Foxp3+ and CXCR5+PD1+ T cells was defined by Flow cytometry as previously described10.
For experiments in PBMCs or monocytes, cells were kept resting for 12–16 h at 4 °C and then replated in appropriate non-tissue culture treated 24 W or 6 W plates at a concentration of 250,000 cells/cm2/250 μL in immune cells medium before treatment and stimulation.
For differentiation of monocytes-derived macrophages (MDM), monocytes were directly plated in tissue culture treated 6 W plates at the same concentration in immune cells medium supplemented with 1% sodium pyruvate and 50 ng/mL human M-CSF (macrophage colony-stimulating factor, Peprotech #300-25) then differentiated for 7 days with medium replenishment every 2 days.
For TLR stimulation of PBMCs, monocytes or differentiated MDM, cells were pre-treated with indicated doses of GV101 or KD025 for 1.5 h in fresh medium. Cells were then stimulated with the TLR4 agonist LPS at 100 ng/mL (Sigma Aldrich #L6011). 24 h after stimulation, cells supernatants were collected, secreted cytokines were measured by ELISA and cells were lysed in Tris 50 mM pH 7.9, 300 mM NaCl and 1% Triton X-100 for preparation of whole cell extracts and western blot analysis.
Culture and stimulation of human and murine primary Kupffer cells
Frozen and previously characterized human primary Kupffer cells were obtained from Sekisui/Xenotech (#HK1000.H15). At the time of the study, human Kupffer cells were available from 5 donors: #H1292 (male donor, used for 2 independent repeats), #H1294 (male donor, used for 3 independent repeats), #H1467 (female donor, used for 1 independent repeat), #H1468 and #H1469 (male donors, each used for 1 independent repeat). Cells were thawed, plated in tissue-culture treated 48 W plates at a concentration of 125,000 cells/cm2/250 uL in OptiThaw Kupffer Cell medium (Sekisui/Xenotech #K8700), and cultured for 10 days with medium replenishment every 3 days. Cells were pre-treated with indicated doses of GV101 or KD025 for 1.5 h in fresh medium. Cells were then stimulated with the TLR4 agonist LPS (Sigma Aldrich #L6011) or the TLR2/TLR6 heterodimer agonist Pam2CSK4 (Invivogen # tlrl-pm2s-1), both at 1 ug/mL, for 24 h before collection of supernatants, analysis of cytokines secretion by ELISA, and preparation of whole cell extracts. ELISA data represent 5-8 independent repeats from the 5 available Kupffer cells donors. For Western Blot analysis, and due to limited amount of cells for each sample, cells from donors #H1467, #H1468 and #H1469 were pooled to reach a sufficient amount of proteins in the whole cell extracts.
Murine primary Kupffer cells were obtained from Glow Biologics (#GBP-1758KC). At the time of the study, cells were available from 2 mice/lots: #14162122 and #17392123. Cells were plated and stimulated as detailed for human Kupffer cells, using immune cells medium.
Pre-Adipocytes culture and adipogenesis assays
Human subcutaneous pre-adipocytes (SP-F-1) and murine 3T3L1 (SP-L1-F) cells were purchased from ZenBio Inc. Cells were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL of penicillin, 100 μg/mL of streptomycin and 1xAmphotericin B according to manufacturer’s instruction. For human cell adipogenesis induction, cells were plated at confluence for overnight, the following day, cells were fed with adipocyte differentiation medium (DM-2, ZenBio) with or without indicated concentrations of ROCK inhibitors, 7 days after induction, cells were subjected to Oil Red O staining, or collected for RNA extraction, or collected for western analysis. For murine 3T3L1 cell adipogenesis, cells were plated at confluence, after two days, cells were fed with differentiation medium (DM-2, ZenBio) with or without indicated concentrations of ROCK inhibitors, incubated for 3 days, then cells were switched to adipocyte maintenance medium (AM-1, ZenBio) with or without indicated concentrations of ROCK inhibitors for 3 more days. 7 days after induction, cells were subjected to Oil Red O staining, or collected for RNA extraction, or collected for western analysis.
Oil Red O staining
Cells were washed with 2xPBS, fixed with 10% formalin for 1 h, then washed with 2 x H2O and 1 × 60% isopropanol. After washing, cells were stained with Oil Red O for 20 min. After staining, cells were washed with 5 x H2O, and ready for viewing under microscope. For Oil Red O quantification of lipid accumulation, Oil Red O-stained cells were washed with 3 × 60% isopropanol, then stained Oil Red O were extracted by applying 100% isopropanol to the cells for 10 min. Intensity of extracted Oil Red O was measured by the absorbance at 492 nm.
Quantitative real-time RT-PCR
Total RNA was purified using RNeasy Plus kit (Qiagen). 10–500 ng RNA was subjected to first-strand cDNA synthesis using Maxima™ H Minus cDNA Synthesis Mastermix (Thermo Scientific). Gene expression was analyzed by quantitative real-time PCR in 20 μl reactions with SYBR Green (Cat#4367659, Applied Biosystems by Thermo Fisher Scientific). All gene expression levels were first normalized with 18 s RNA as an internal control and then were normalized with the level of vehicle (DMSO) treated cells. Primers used in real-time PCR are as following: 18 S, forward, agtccctgccctttgtacaca, reverse, cgatccgagggcctcacta; human Glut4, forward, TCTTCGAGACAGCAGGGGTA, reverse, CTCCACCAACAACACCGAGA; mouse Glut4, forward, GCCCCACAGAAGGTGATTGA, reverse, GAAGATGGCCACGGAGAGAG.
MRC-5 cell culture and assays
Human lung fibroblast MRC-5 cell (CCL-171) was purchased from American Type Culture Collection (ATCC) and was cultured in EMEM containing 10% fetal bovine serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin according to manufacturer’s instruction. On day 1, cells were seeded in 24-well plates at density of 50,000 cell/well in 1 ml culture medium; On day 2, cells was switched to starve EMEM containing 0.5% FBS; On day3, cells were treated with indicated concentration of ROCK inhibitors, incubated for 1 h, and then TGF-β1 (2.5 ng/mL) was applied to the cells; On day 5 (48 h after treatment), cultured medium was collected for Col1α1 ELISA (human Pro-Collagen 1α1 DuoSet ELISA kit, R&D systems), cells were collected for RNA extraction or for western analysis. Primer’s sequence used for real-time PCR are as following: αSMA, forward, CCCAGACATCAGGGGGTGAT, reverse, TCGGGTACTTCAGGGTCAGG; CTGF, forward, CGCACAAGGGCCTATTCTGT, reverse, GAGCACCATCTTTGGCGGT; Col3A1, forward, CAGCCTCCAACTGCTCCTAC, reverse, CCAGGGTCACCATTTCTCCC.
siRNA Knock down in Human subcutaneous pre-adipocytes
ON-TARGETplus SMARTpool siRNA that specifically targeting ROCK1 (L-003536-00-05), ROCK2 (L-004610-00-05) and control siRNA (D-001810-10-05) were purchased from Dharmacon (Thermo Fisher Scientific Inc.). Transfection was performed using Amaxa™ P1 Primary Cell 4D-Nucleofector™ X kit L with Amaxa™ 4D-Nucleofector™ X unit (Amaxa Biosystems, Lonza) according to manufacturer’s instructions. Briefly, cells were trypsinized from culture plates, 5 × 105 cell were used for each transfection in 100 µl Single Nucleocuvette. After transfection, cells were plated and recovered for 6 h in incubator before changing to culture medium. Cells were collected for Western blotting analysis 48 h after transfection.
Preparation of whole cell/tissue extracts and Western blots
At the time of collection, PBMCs and monocytes cultures were harvested, while MDM were gently scraped in cold PBS after medium removal. Cells or homogenized tissues were centrifuged at 400 g for 5 min at 4 °C, and cell pellets were lysed in 50 mM Tris pH 7.9, 300 mM NaCl and 1% Triton X-100 supplemented with protease and phosphatase inhibitors cocktail (ThermoScientific #A32961) for 15 min on ice. After centrifugation at 17,000 g for 10 min at 4 °C, whole cell extracts were collected, protein concentrations were determined using Bradford method (ThermoScientific #23238) and samples were reduced and denatured in DTT-containing XT Sample buffer (Biorad #1610791) for 10 min at 95 °C.
Proteins were resolved by SDS-PAGE (Invitrogen mini gels system) and transferred onto nitrocellulose membranes (Invitrogen iBlot 2) before blocking with 10% evaporated milk in PBS 0.2% Tween and incubation with the corresponding primary and HRP-conjugated secondary antibodies, all diluted in PBS 0.2% Tween 10% evaporated milk at concentrations recommended by manufacturer. Signals were visualized using the Amersham ECL, Pierce ECL Plus or SuperSignal West Pico Plus ECL detection reagents and imaged using Invitrogen iBright CL1500 imager. Red Ponceau staining was used before immunoblotting to check for transfer efficiency. All blots were probed for β-actin as a loading control. When indicated, Western Blot quantification was performed using Fiji/ImageJ software.
For Western blots, primary and HRP-conjugated secondary antibodies were obtained 1) from Cell Signaling Technology: anti-β-Actin (4970), anti-Akt (2920), anti-Phospho-Akt S473 (4060), anti-AMPK (2793), anti-Phospho-AMPK T172 (50081), anti-Cofilin S3 (5175), anti-Phospho-Cofilin (3313), anti-p70 S6K (34475), anti-Phospho-S6K T389 (9205), anti-mTOR (2972), anti-Phospho-mTOR S2448 (2971), anti-α-Smooth Muscle Actin (SMA; 56856), anti-STAT3 (12640), anti-Phospho-STAT3 Y705 (9145), anti-Phospho-STAT5 Y694 (4322), HRP-conjugated anti-Rabbit IgG (7074), and HRP-conjugated anti-Mouse IgG (7076); 2) from Sigma Aldrich: anti-ROCK1 (HPA007567) and anti-ROCK2 (HPA007459); or 3) from eBioscience: CD185 (CXCR5) (12-1859-42), CD279 (PD-1) (11-9969-42), Foxp3 (12-4777-42), CD4 (11-0049-42) and Fixable Viability Dye (65-0866-14).
Measurement of cytokines concentrations was performed using ELISA kits or antibodies 1) from R&D Systems: hCXCL13 (DY801), hIL-1b (DY201), hIL-10 (DY217B), hIL-17 (DY317), hIL-23 (DY1290), hIL-6 (DY206), hPro-Collagen1α1 (DY6220), hTNF (DY210), mIL-6(DY406), mTNF(DY410) or 2) from eBioscience: hIL-21 capture (14-7219-82) and detection (13-7218-81) antibodies, according to the manufacturer’s recommendations. Briefly, 96 W Immulon plates were coated overnight with respective capture antibodies diluted at the recommended concentrations in PBS, washed 4 times and blocked with PBS 5% BSA for 1 h before adding the cell supernatants either pure or diluted in appropriate cell medium. Serial dilutions of recombinant cytokines were made on each plate to serve as concentration standard. Incubation with supernatants was done for 2–3 h at RT or overnight at 4 °C with gentle shaking. Plates were then washed 4 times with PBS 0.2% Tween before incubation with respective detection antibodies for 2–3 h at RT with gentle shaking. After washing 4 times, reaction was initiated by adding Ultra-TMB (ThermoScientific #34029), stopped adding a solution of 0.18 M H2SO4, and absorbance at 450 nm was measured using 540 nm as reference absorbance. Exact cytokine concentration was calculated by using standard curve. When indicated, cytokine concentrations were normalized to the condition with stimulus alone (set at 100) to compare independent samples purified from independent blood donors.
Statistical analysis and reproducibility
Different statistical tests were performed to analyze experiments, as indicated: Unpaired t-test or one way ANOVA followed by multiple comparisons Sidak tests to allow two-by-two comparisons. Significance is indicated on each figure as follows: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. All in vitro data represent at least 3 repeats unless otherwise stated.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.