Clinical samples
Subjects with an MM genotype were selected from the Alpha‐1 Foundation DNA and Tissue Bank for use as a control group. Study subjects were chosen from a trial based at the University of Florida that followed patients with AATD over a 3-year period to evaluate progression of liver disease (Table 1). Plasma from subjects with a ZZ genotype and liver biopsies indicating presence of fibrosis and high levels of PASD positive cells were used (7). All AATD individuals confirmed by genetic testing who were age 21–71, from the US or Canada, and willing to consent to a liver biopsy were eligible. Decompensated liver disease (ascites, variceal bleeding, hepatic encephalopathy, hepatocellular carcinoma) was excluded. Recipients of a liver or lung transplant were excluded. A total of 26 patients were included. All participants gave informed, written consent prior to enrolling. The study was approved by the University of Florida institutional IRB (13). All authors had access to the study data and reviewed and approved the final manuscript.
Cell culture
Human LX-2 cell line, purchased from MilliporeSigma (Burlington, MA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% FBS and 1% PrimocinTM from InvivoGen (San Diego, CA), at 37 °C in a humidified incubator under 5% CO2. iPSC-derived HSCs were differentiated from induced pluripotent stem cells (iPSCs) (15). Briefly, peripheral blood mononuclear cells-derived iPSCs provided by iPSC core at the University of Florida were incubated with BMP4 from StemCell Technologies (Vancouver, BC) for 4 days to induce mesodermal progenitors. Following this step, the cells were subsequently incubated with BMP4, FGF1, and FGF3 until day 6 to induce a liver mesenchymal submesothelial phenotype. From days 6 to 8, the cells were incubated with FGF1, FGF3, retinol, and palmitic acid, from StemCell Technologies (Vancouver, BC). The final step of differentiation was modeled by incubation of the cells with retinol and palmitic acid from day 8 to day 12. We next characterized differentiated iPSC-HSCs on day 12 (Supplementary figure 1a and B). iPSC-HSCs were analyzed for the expression levels of markers associated with HSCs (Supplementary figure 1C) including TIMP (Tissue Inhibitor of Metallopeptidase Inhibitor 1), CoL1A1(Collagen type1 Alpha1), -SMA (Alpha Smooth Muscle Actin) and LRAT (Lecithin: Retinol Acyl Transferase).
Isolation of EVs
EVs were isolated from approximately 3 mL of cryopreserved plasma by differential centrifugation (16). Briefly, the plasma was diluted in PBS (1:1) and centrifuged at 2,000 x g for 30 minutes at 4°C and at 12,000 rpm for 30 minutes at 4°C. Supernatants were collected into ultracentrifuge tubes and centrifuged in a Beckman SW-40Ti swinging rotor ultracentrifuge (Beckman Coulter) at 110,000 x g for 2 hours at 4°C. Resuspended pellets in PBS were filtered through a 0.22-μm filter (Millipore, Billerica, MA) and centrifuged at 110,000 × g for 70 minutes at 4°C. EV pellets were washed with PBS and centrifuged at 110,000 × g for 70 minutes at 4°C and resuspended in 200 μL of PBS. EV preparations were conserved at −80 °C for later use.
Particle number and size measurement
The size distribution and concentration of the isolated EVs was analyzed by Nanosight NS300 system (Nanosight and Malvern, United Kingdom). Briefly, purified EVs were homogenized by vortexing followed by dilution of 1:100 in sterile PBS. Each sample analysis was conducted for 60 seconds. Data was analyzed by Nanosight NTA 2.3 Analytical Software with the detection threshold optimized for each sample and screen gain at 10 to track as many particles as possible with minimal background. A blank 0.2 μm- filtered 1x PBS was also run as a negative control. At least five analysis were done for each individual sample.
Transmission electron microscopy
Purified EVs were fixed with 2% paraformaldehyde. A 20 μl drop of the suspension was loaded onto a formvar coated grid, negatively stained with 2% aqueous uranyl acetate, and examined under a Hitachi 7600 transmission electron microscope (Hitachi High-Technologies, Schaumburg, IL) equipped with a Macrofire monochrome progressive scan CCD camera (Optronics, Goleta, CA) and AMTV image capture software (Advanced Microscopy Techniques, Danvers, MA).
EVs RNA Isolation, small RNA Library Preparation, Sequencing, and Bioinformatics
RNA was isolated using exoRNeasy Maxi kit (Qiagen) from purified EV fractions derived from 4 MM healthy and 5 ZZ plasma samples. The quantity and quality of the RNA were determined using the Agilent RNA 6000 Pico Kit and a Small RNA Kit Chip on the Agilent Bioanalyzer instrument (Agilent Technologies). Sequencing libraries were constructed using ∼2 ng of total EV RNA with Small RNA Library Prep Set for Illumina kit (New England BioLabs) and were sequenced using Illumina Miseq 1x150 cycles V3 kit at the University of Florida Interdisciplinary Center for Biotechnology Research. The expression of the miRNAs was obtained using miRDeep2 software (14) and differential analysis was performed using the exact test from edgeR package (17). We performed enrichment analysis using Ingenuity Pathway Analysis (IPA) (Ingenuity Pathways Analysis (IPA) system. Redwood, CA: Ingenuity Systems, Inc;) package using the miRNA-mRNA target link module to link the discovered significantly differentially expressed miRNAs with their mRNA target genes. Because IPA does not include prediction scores, to validate the miRNA target genes, we performed additional computational analysis using TargetScan (Release 7.1. Jun, 2016.) to identify target genes with good predicted scores. In addition, we used IPA to identify miRNA-mRNA target genes which have been experimentally confirmed.
Real-time qPCR validation of selected miRNAS from Small RNA-sequencing
Reverse transcription reactions were performed using the TaqMan Advanced miRNA Assays kit (Applied Biosystems. Inc, CA; USA) using 2 µL of cell-free RNA extracted from exosomes. Real-time PCR reactions were performed in duplicate in 20μL reaction volumes using 10 μL SensiFAST Probe Lo-ROX 2x PCR Master Mix (Bioline, Memphis, TN), 1 μL TaqMan Advanced miRNA assay (20x) (Applied Biosystems. Inc, CA; USA), 4 μL of nuclease free water and 5 μL of RT product after a 1:10 dilution. Real-time PCR was carried out on an Applied BioSystems 7500 Fast thermocycler (Applied Biosystems. Inc, CA; USA) programmed as follows: 95 °C for 20 seconds followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 seconds. We used hsa-miR-21-5p and hsa-miR-26a-5p, two of the most stable miRNAs in terms of read counts, as a endogenous controls. All fold-change data were obtained using the delta-delta CT method (2−ΔΔCT) 28. Five differentially expressed miRNAS detected after small RNA-seq were validated using RT-qPCR (hsa-miR-128-3p, hsa-miR-6809-5p, hsa-miR-125a-5p, hsa-miR-151a-5p and hsa-miR-99a-5p).
Cytokine measurement
We first identified priority cytokine candidates based on known liver fibrosis pathophysiology and previously published literature, then selected from the assays available at Myriad-RBM, which were primarily multiplexes (Luminex, Myriad-RBM Inc., Austin TX). Then purified EVs from 20 MM and 20 ZZ plasma samples (Table 1), were suspended in a mixture of water and PBS (1:2). EVs in solution were lysed using an equal volume of IP lysis buffer. Lysed EV samples to which Triton X was added at final concentration of 1% were run on the MilliPLEX Human High Sensitivity T Cell Magnetic Bead Panel Luminex kit for measurement of 21 unique cytokines per plex according to manufacturer’s instructions (EMD Millipore, St. Louis, MO).
Labeling of EVs and uptake
Purified EVs from plasma (109 CD63-positive particles) were incubated with Fast DiO membrane dye (Invitrogen) at a final concentration of 2 μg/mL for 1 hour at room temperature. The purification process of washing and ultracentrifugation was repeated twice before the labeled EV pellet was resuspended in PBS (18). For microscopic analysis, LX2 cells were incubated with DiO-labeled EVs for 1 hour at 37°C. After incubation, cells were washed with PBS to remove unbound labeled EVs and subsequently imaged with a Keyence fully motorized BZ-X800 microscope (KEYENCE America, Chicago, USA).
For flow cytometry analysis, LX2 cells were seeded in six-well plates and grown overnight and DiO-labeled EVs (2×107) in 100 μL PBS/well (CD63-positive) were added and incubated at 37°C for 1 hour. Cells stained directly with 1 μL/well DiO (1 μg/mL) served as a positive control, and unstained cells as a negative control. Cells were analyzed using a Beckman Coulter Gallios Flow Cytometer using Kaluza acquisition software and Flow Jo for analysis. Each experimental group was performed in triplicate.
Co-incubation of human plasma-derived EVs with hepatic stellate cells
HSCs were plated at the cell concentration of 200,000 per well in 12-well plates. Isolated EVs (pooled from 5 control or AATD individuals (isolated from 250 mL plasma) were added to each well and the plate was incubated for 48 h at 37 °C (16). HSCs were incubated with the same volume of PBS and have been used as negative control.
Cell migration and proliferation assay
Cell migration was assessed by measuring the repair of a linear wound generated in the confluent monolayer of cells. Hepatic stellate cells were (LX-2) plated at an equivalent density on chamber slides (Lab-Tek, Westmont, IL), and incubated with or without plasma derived EVs for 4 hours before the application of a linear scratch in cell monolayers using a sterile plastic pipette tip. Cell migration was recorded for 4, 8, 16 and 24 h using an Applied Precision deconvolution microscope. To assess the proliferation rate of the cells, 4 different fields of each condition has been subjected to cell counting for 4, 8, 16 and 24 h and the mean values have been recorded as the total number of cells.
Quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Takara, A7603-1), and reversely transcribed through SuperScript VILOTM kit (Invitrogen, Carlsbad, CA). Real-time PCR analyses were performed with SensiFAST qPCR Master Mix (Bioline, Memphis, TN) on a 7500 Real-time PCR system, Applied Biosystems. Probes used for human Acta2 (Hs00426835_g1), Col1a1 (Hs00164004_m1), Timp1 (Hs01092512_g1) and LRAT (Hs00428109_m1) detection were purchased from Thermofisher (Carlsbad, CA).
Immunoblotting
LX-2 cells and iPSc-derived HSCs were seeded at 3 x 105/well in 6-well plates with or without MM or ZZ plasma-derived EVs for 24 hours. Protein levels in the cell lysate homogenates utilizing RIPA buffer were determined using the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL). Rabbit polyclonal antibodies were used to detect total and phospho-IKK (19), p65, p50 (20), total and phospho-STAT (21), total and phospho-JAK (21) and GAPDH (Cell Signaling, Danvers, MA), CD81 (22), CD63 (23) and TSG101 (24) (Proteintech, Chicago, IL). Proteins were detected by using a Super Signal West Dura Extended Duration Substrate Kit from Thermo Scientific.
Immunostaining and immunofluorescence microscopy
LX-2 cell line treated with or without MM or ZZ plasma-derived EVs were grown on glass coverslips. After 24 hours, the cells were fixed with 4% paraformaldehyde in PBS for 20 minutes. The cells were incubated for 1 hour with blocking buffer at room temperature, followed by incubating overnight at 4°C with primary antibodies (1:400). The cells were washed with 1X PBS and incubated for 1 hour with secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG). Images were collected using a Keyence fluorescence microscope (Osaka, Japan). Images were processed for brightness and contrast and filtered for noise following good practices as outlined by Rossner and Yamada.
Histology, histochemistry and immunohistochemistry
Paraffin-embedded liver tissue sections were routinely de-paraffinized with xylene and a graded series of ethanol. Some tissue sections were stained with hematoxylin–eosin (HE) for simple morphological examination. For immunostaining on paraffin‐embedded liver tissues, paraffin blocks were sliced into 5 μm sections, deparaffinized with xylene, and rehydrated with decreasing concentrations of ethanol in water. Antigen retrieval was achieved by incubation for 20 minutes in hot (95°C) sodium citrate buffer (pH 6.0) and 20 minutes of cooling at room temperature. Endogenous peroxidases were quenched by incubation in 3% hydrogen peroxide for 20 minutes. Sections were washed with PBS. Primary antibodies were applied for 60 minutes at room temperature in a humidified chamber. After rinsing the slides in PBS, they were incubated in secondary antibody for 1 hour at room temperature. After washing with PBS, slides were incubated with Vectastain ABC (Vector Laboratories) for 30 minutes. After washing with PBS, color development was achieved by applying diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories) for two to five minutes. Images were acquired and the signal intensity was quantified using a Keyence BZ-X700 microscope.
Plasma levels of CRP quantification
Plasma samples were centrifuged at 20,000 x g for 10 minutes to remove fibrin or debris. Samples (100 µL) were then loaded undiluted and CRP levels measured using the Cardio Phase hsCRP assay on a Siemens Nephelometer calibrated with the Rheumatology Standard according to manufacturer’s directions. The assay has a range of 0.1 - 50 mg/L.
Plasma levels of CXCL10 quantification
CXCL10 levels were measured in the plasma of ZZ patients with liver disease and MM controls using an ELISA kit from R&D Systems according to manufacturer’s instruction. Samples were diluted 1:2 in assay diluent before being applied to the assay plate for 2 hours incubation at room temperature. The plate was washed, Human IP-10 Conjugate added to each well, and the plate incubated for another 2 hours at room temperature. The plate was washed again and substrate solution added to each well. The plate was incubated for 30 minutes in the dark before adding stop solution. The optical density was measured using a SpectraMax spectrometer at 450 nm and the sample concentrations calculated based on the standard curve.
Liver histology
A percutaneous liver biopsy was performed with a 16 gauge BioPince™ core biopsy needle after using ultrasonography to mark the location. The sample was fixed in formalin and processed for examination. Stains included H&E, trichrome, PAS/PAS + D and Prussian blue. Portal inflammation and hepatocyte degeneration were noted. (7).
FibroScan and ultrasound
Liver stiffness measurements (LSM) were performed by using FibroScan device powered by vibration controlled transient elastography (Echosens). Two individuals performed the procedure after training on proper technique. LSM were considered reliable if the following three criteria were met: at least 10 valid measurements, ≥60% success rate, IQR/median ≤30% (13)
Data Analysis
We extracted data on sample size, mean cytokine concentration, standard deviation (SD), and p-value to calculate the effective size (ES). The sample size, mean cytokine concentration, and SD were used to calculate the ES. Two-sample t test for mean difference with unequal variance was used to test the power of each group sample size of 20 using the usual significance level alpha = 0.05. Power analysis and statistical analyses were performed using SAS and GraphPadPrism 8 software (San Diego, CA). If cytokine concentration data were not available, the ESs were generated by sample size and p-value. All results are expressed as mean ± S.E. Statistical analyses were performed using Prism 8 by Student t-test or Mann-Whitney U test. Values of P < 0.05 was considered statistically significant. power analysis showed the power of > 0.842.