Cirrhosis is the consequence of many forms of chronic liver diseases and is characterized by replacement of liver tissue by fibrosis, scar tissue, and regenerative nodules. Liver transplantation, which often remains the only viable treatment option, is only available for a fraction of patients in need, mainly due to the growing demand for transplants in view of an increasing shortage of donor organs. Therefore, there is an urgent need for antifibrotic treatments, which can prevent, halt, or even reverse advanced fibrosis.
In recent years, significant progress has been made in our understanding of fibrosis in general and liver fibrosis in particular. Liver fibrosis can be viewed as a dynamic process, characterized by a preponderance of extracellular matrix (ECM) production, i.e., fibrogenesis, over its degradation, i.e., fibrolysis, which finally leads to distortion of the hepatic architecture (cirrhosis) and loss of organ function.
In hepatic fibrosis, the excessive ECM is produced by activated mesenchymal cells which resemble myofibroblasts. They derive from quiescent hepatic stellate cells (HSC) and periportal or perivenular fibroblasts, here collectively termed HSC. Activation of HSC by several profibrogenic cytokines and growth factors, especially by TGF-β1, is a general feature of fibrosis progression. These factors are mainly produced by activated macrophages or cholangiocytes, but also by liver infiltrating lymphocytes, as shown recently for CD8+ T cells.
Activated HSC can also release pro-inflammatory chemokines/cytokines that attract and activate inflammatory cells, such as MCP-1, IL-6, and TGFβ1. Furthermore, a proinflammatory milieu, e.g., via TNFα and INFγ, can induce adhesion molecules on HSC that further attract inflammatory cells, such as CD54 (ICAM-1) or VCAM-1, the expression of chemokines like CXCL9 and CXCL10, and of chemokine receptors like CXC3R1.
Several studies suggest that even advanced experimental and, possibly, human liver fibrosis can regress once pathogenic triggers are eliminated and sufficient time for recovery is available. Interestingly, the same cells that drive fibrogenesis (HSC) can become major effectors of fibrolysis, e.g., via production and activation of certain matrix metalloproteinases (MMPs). This has been shown in vitro when dermal fibroblasts are plated from a 2D cell culture dish into a 3D collagen gel. Thus under 3D conditions activated fibroblasts/myofibroblasts contract and upregulate MMP production, while procollagen I, the major component of scar tissue is downregulated. However, relevant triggers of myofibroblast or HSC fibrolytic activation remain largely unknown.
One study suggests lymphocytes can modulate fibroblasts in a different, non-cytokine mediated manner. A crude microparticle (MP) preparation released from membranes of Jurkat T cells (an immortal lymphoma T cell line) during activation and early apoptosis could induce synovial fibrolytic MMP expression in fibroblasts. However, it remains unclear how these MP exerted their fibrolytic effects.
In one aspect, the invention features a method of diagnosing a subject for an inflammatory disease (e.g., hepatitis, hepatitis C, non-alcoholic steatohepatitis, celiac disease, inflammatory bowel disease, and other inflammatory diseases) by determining the amount of microparticles derived from T cell and other inflammatory cell subsets, including but not limited to CD4+ and/or CD8+ T cells, iNKT cells, CD14+ monocytes/dendritic cells, CD15+ neutrophils, or CD41+ platelets in a blood sample of the subject, where an elevated amount of microparticles derived from these cells diagnoses the subject as having a disease that is dominated or influenced by one or more of T cells, iNKT cells, CD14+ monocytes/dendritic cells, CD15+ neutrophils, or CD41+ platelets.
In the foregoing aspect, the method can further include isolating the microparticles from the blood sample prior to determining the amount of microparticles, e.g., derived from CD4+ and/or CD8+ T cells, iNKT cells, CD14+ monocytes/dendritic cells, CD15+ neutrophils, or CD41+ platelets in the blood.
In any of the foregoing aspects, the determination of the amount of microparticles derived from, e.g., CD4+ and/or CD8+ T cells, iNKT cells, CD14+ monocytes/dendritic cells, CD15+ neutrophils, or CD41+ platelets in the blood sample can include measuring the amount of CD4+ and/or CD8+ cell, iNKT cells, CD14+ monocytes/dendritic cells, CD15+ neutrophils, or CD41+ platelets microparticles in the blood sample or isolated microparticles (e.g., by contacting the blood sample with antibodies to CD4 and/or CD8, Valpha24Vbeta11, CD14, CD15, or CD41).
In another aspect, the invention features a kit for diagnosing an inflammatory disorder including at least one binding agent (e.g., an antibody or
antibody fragment) and instructions for measuring the amount of microparticles derived from particular cell types in a blood sample of the subject, where the amount of microparticles derived from particular cell types diagnoses the subject as having an inflammatory disease associated with said particular cell type. The at least one binding agent including a binding agent specific for one or more of the following cell types: CD4+ and/or CD8+ T cells, CD14+ monocyte or dendritic cells, invariant chain natural killer (iNKT) T cells, and/or CD41+ platelet cells
In another aspect, the invention features a pharmacological composition including insolated microparticles (e.g., synthetic microparticles, microparticles isolated from a human, or microparticles isolated from a T cell line like Jurkat cells), wherein the microparticles include receptors or membrane bound molecules found on CD4 and/or CD8 T cells and in addition or in the alternative include CD54 and/or CD147 receptors (e.g., recombinant receptors).
The foregoing pharmaceutical compositions can further include siRNA against at least one gene selected from the group consisting of procollagens I, III, IV, V, VI, HSP47, TGF beta1, TGFbeta2, PDGF-B, CTGF, TGF beta receptors I, II and III, PDGFbeta receptor, integrins alpha1beta1, alpha2beta1, alpha3beta1, alpha5betal, MCP-1, CXCL4, CCL2, and CXCR2.
In another aspect, the invention features a method of treating liver fibrosis in a subject by administering any of the foregoing pharmaceutical compositions.
Included is also the isolation and expansion of autologous or heterologous (other donor than the patient) T cells from peripheral blood, e.g., via CD3 (CD8) affinity chromatography, negative selection or other standard T cell isolation procedures, followed by PHA, cytokine driven or other standard nonspecific or specific in vitro T cell expansion methods, with the aim of generating and purifying of large numbers of homogeneous MP in vitro that will be then infused into the patient as therapy. This method will take advantage of autologous tissue histocompatibility to reduce any potential side-effects and will allow repeated treatments.
By “blood sample” is meant a blood, serum, or plasma specimen obtained from a patient or a test subject.
By “treating” is meant administering a pharmaceutical composition for prophylactic and/or therapeutic purposes or administering treatment to a subject already suffering from a disease (e.g., fibrosis of the liver) to improve the subject's condition or to a subject who is at risk of developing a disease. In the case of liver fibrosis, treatment would result in an increase (e.g., by at least 5%, 10%, 25%, 50%, 75%, 100%, 200%, 500%, or more) in fibrolysis or a reduction (e.g., by at least 5%, 10%, 25%, 50%, 75%, or more) of overall fibrosis or fibrogenesis, or fibrosis or fibrogenesis in a particular region of the organ.
By “elevated” is meant an amount of MP in a sample that is at least 5%, 10%, 25%, 50%, 75%, 100%, 200%, 500%, or more, greater than that measured in a control sample (e.g., from a healthy subject).
By “decreased” is meant an amount of MP in a sample that is at least 5%, 10%, 25%, 50%, 75%, or less, than that measured in a control sample (e.g., from a healthy subject).
By “inflammatory disease” is meant a disease characterized by specific T cell, dendritic cell/monocyte (CD14+), neutrophil (CD15+) or platelet (CD41+)) responses.
“ST” refers to 0.04 μM/mL staurosporine, and “MP” refers to S10-MP or S-100-MP from apoptotic Jurkat T cells suspended in 350 μL medium for 24 hours.
In general, the invention features methods of diagnosing the overall inflammatory activity and profile of inflammatory diseases (e.g., hepatitis C or NASH, as well as of other diseases that are characterized by a specific T cell, or dendritic cell/monocyte (CD14+), neutrophil (CD15+) or platelet (CD41+)) response, based on the relative presence of the respective microparticles (MP). The invention also features methods of decreasing fibrosis in the liver by administering CD8+ (CD4+) MP to subjects with liver fibrosis. The invention is based on the discovery that blood samples taken from subjects with, e.g., hepatitis C, NASH, celiac disease, IBD (and other diseases characterized by significant inflammation and T cell turnover) contain characteristically elevated (or decreased) levels of CD4+, CD8+, iNKT, CD14, or CD15 MP. Further, administration of CD8+ MP is effective to induce fibrolysis in in vitro models of liver fibrosis. The proposed mechanisms are schematically illustrated in
The invention features methods of diagnosing diseases based on the presence and features of MP in subject samples (e.g., blood samples). MP bear the cell surface receptors of the blood cells (e.g., T cells) from which they derive. Therefore, the presence of MP with certain surface markers is indicative of the disease and importantly of cell specific disease activity with which the corresponding blood cell (e.g., T cell) is associated.
For example, MPs with CD4 and CD8 markers are diagnostic of hepatitis (e.g., hepatitis C). NASH is also associated with a striking increase in CD14+ (monocyte/dendritic cell) MP. Celiac disease is associated with characteristic changes in CD4+, CD8+ T cell, iNKT and CD41+ (platelet derived) MP. Inflammatory bowel disease is associated with characteristic changes in CD4+ T cell and CD14+ MP.
Diagnosis is based on the relative frequency of MPs in a subject sample (e.g., a human, mouse, rat, dog, or cat sample) associated with the indicated diseases and the severity and prognosis of the disease can be further ascertained by comparing the MP levels with control levels (e.g., as taken from a healthy subject, or a sample from a subject that, retrospectively, is deemed to have a severe or mild form of the indicated disease).
Diagnosis can be based on the detection of unique MP profiles for patients with chronic hepatitis C (HCV), non-alcoholic steatohepatitis (NASH), various activities of celiac disease, and inflammatory bowel disease (IBD). For example, percentages of CD8 T cell derived S100-MP were elevated in active HCV infection (ALT>100 IU/mL) and NASH, but unchanged in mild HCV infection (ALT<40 IU/ml), in celiac disease and to a lesser degree in successfully treated IBD (the latter two being CD4 T cell dominated diseases as compared to viral hepatitis which is both CD4 and CD8 T cell dominated). Percentages of CD4 T cell derived S100-MP were significantly increased in active HCV infection, NASH and celiac disease. CD41 (platelet-derived) MP were decreased in NASH, celiac disease and IBD, whereas CD15 (neutrophil)-derived S100-MP were non significantly decreased in NASH and celiac disease, but significantly reduced in IBD patients. CD14 (monocyte/dendritic cell-derived) MP were strongly reduced in active HCV infection, mildly increased in IBD and highly increased in NASH. Percentages of invariant chain natural killer (iNKT) T cell (Valpha24/Vbeta11 double positive) derived MP were significantly increased in NASH, celiac and IBD patients. Thus each investigated disease is characterized by an individual pattern of cell specific MP, which can be analyzed by FACS and used as an early diagnostic tool to assess the cellular pattern and intensity of the respective immune activation in the blood.
Measurement of transmembrane proteins (e.g., CD4 or CD8) in MP can be performed directly on a subject sample or upon MP particles isolated from a subject sample. Transmembrane proteins, carbohydrate/glycosaminoglycan/proteoglycan, or lipid/glycolipid/lipoprotein structures can be detected by, for example, contacting the sample or isolated MP with a transmembrane specific antibody (e.g., a fluorescently, peroxidase, streptavidin or luminescent labeled antibody, or a HLA (MHC)-tetramer/pentamer). Antibody (tetramer/pentamer) bound to MP can be detected and quantified, e.g., using FACS analysis, via overall fluorescence or luminescence measurement, or microscopically. MP can also be sorted according to the labeled transmembrane protein, carbohydrate/glycosaminoglycan/proteoglycan, or lipid/glycolipid/lipoprotein structure and subject to quantitative analysis for specific proteins, carbohydrates/glycosaminoglycans/proteoglycans, lipids/glycolipid/lipoproteins, or RNAs and DNAs in the MP.
Methods of isolation MP from subject samples are described herein (e.g., differential centrifugation, antibody or aptamer affinity chromatography with positive or negative selection).
The invention features methods of treating liver disease by administering CD4+ or preferably CD8+ MP (e.g., CD4+ CD8+ MP). These MP can be generated from cells derived from the subject to be treated (e.g., autologous cells) or from other cells and cell lines. Large quantities of MP can be generated ex vivo from T cells by use of agents that activate T cells (e.g., phytohemagglutinin) or induce apoptosis (e.g., UV light, staurosporin, fas ligand, or fas activating antibody).
The treated T cells (e.g., Jurkat cells) can be engineered or further treated to express the desired markers and active principles (e.g., CD54, CD147, antifibrotic/fibrolytic proteins, carbohydrates/glycosaminoglycans/proteoglycans, lipids/glycolipid/lipoproteins, or RNAs and DNAs). Such expression can be obtained through, e.g., the introduction of recombinant constructs. In one embodiment, the Jurkat cells are not activated prior to induction of MP formation.
Therapy according to the invention may be performed alone or in conjunction with another therapy and may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment optionally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed, or it may begin on an outpatient basis. The duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient responds to the treatment.
Routes of administration for the various embodiments include, but are not limited to, topical, transdermal, nasal, and systemic administration (such as, intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, or oral administration).
Indications for the therapy of the invention includes any liver disease associated with liver fibrosis including cirrhosis of the liver due to alcohol consumption, hepatitis associated with viral infection, nonalcoholic steatohepatitis, autoimmunity, haemochromatosis, congenital, immune mediated or acquired disease, Wilson's disease, cystic fibrosis and other genetic or congenital biliary and nonbiliary liver diseases, post transplant fibrosis, Budd-Chiari syndrome, and hepatocellular carcinoma.
T Cell Derived Microparticles Circulate in the Blood Plasma of Healthy Controls and are Increased in Patients with Active Hepatitis C
We searched for T cell derived microparticles (MP) in human plasma from normal controls and patients with chronic hepatitis. Using a two-step centrifugation at 10,000 and 100,000 g, we focused on S100-MP. FACS analysis using the MP marker Annexin V and the general T cell marker CD3 showed that indeed T cell derived MP were present in human blood plasma (
Due to the low numbers of circulating MP, initial characterization and functional analyses were performed with T cell MP generated from the human Jurkat T cell line (that expresses CD4) and from peripheral blood T cells of healthy human donors. We stimulated MP release either by activation using phytohemagglutinin (PHA), or by induction of apoptosis using the tyrosine kinase inhibitor staurosporine (ST). The S10-MP fraction was Annexin V-low and CD3-low, and the S100-MP fraction was Annexin V-high and CD3-high (
CD3 T Cell Receptor Transfer from S100-MP to Cell Membranes of Human Hepatic Stellate Cells
The exclusive expression of transmembrane CD3 on T cells allowed us to monitor the transfer of CD3 (and likely other transmembrane molecules) from S100-MP to human LX-2 hepatic stellate cells (HSC). FACS analysis demonstrated that after six hours of incubation with S100-MP, the transfer of CD3 from MP to HSC peaked, with 17% of the HSC being positive for CD3 (
Fibrosis related transcripts were measured from 200×103 serum-starved human LX-2 HSC 24 hours after addition of 1×103 or 50×103 S100-MP from Jurkat T cells using quantitative RT-PCR. S10-MP, plain medium, and ST alone served as controls. MP were obtained from PHA-activated and/or apoptotic (ST-treated) Jurkat T cells. After T cell apoptosis induction, significant changes in fibrosis-related transcripts were found with 50×103 S100-MP, while equivalent amounts of S10-MP had no effect (
It was reported earlier that MP derived from macrophages could trigger apoptosis in recipient cells. Since it is known that matrix metalloproteinases (MMPs), especially MMP-3 is upregulated in cells undergoing apoptosis and since our data show that indeed S100-MP derived from apoptotic T cells prominently upregulated MMP-3 in HSC, we evaluated apoptosis induction by S100-MP using Annexin V externalization and 7-amino-actinomycin D (AAD) labeling as readout. Jurkat T cell-derived 5100-MP did not induce enhanced apoptosis or necrosis in HSC after 24 hours of incubation, whereas HSC that were treated with ST alone exhibited up to 14% necrosis and 10% late apoptosis (
Human HSC were exposed to 5 ng/mL TGFβ1, which elicits a strong fibrogenic response. Jurkat T cell-derived S100-MP did not only blunt the TGFβ1 response, by reducing procollagen α1(I) expression, but even induced fibrolytic MMP transcripts beyond the levels produced by unstimulated HSC (
Comparison of the Effect of S100-MP Derived from CD4+ and CD8+ T Cells
S100-MP were produced and purified from peripheral T cells of healthy donors. Overall, apoptotic CD4+ T cell-derived MP induced MMP expression in HSC much less efficiently than MP from CD8+ T cells, irrespective of their mode of generation (with or without prior activation by PHA). Thus MP from CD4+ T cells did not significantly affect MMP-1, -3, -9, -13, TIMP-1 or procollagen α1(I) expression. If MP shedding was induced only by CD4+ T cell activation with PHA, a significant induction was observed for MMP-1, MMP-3, and MMP-9 mRNA (between 1.7- and 3-fold), while procollagen α1(I) and TIMP-1 transcript levels remained unchanged (
It remained to be shown what cell membrane molecule(s) or receptor(s) mediate(s) attachment and uptake of S100-MP by HSC. CD54 is expressed by HSC and upregulated by proinflammatory signals. Our FACS analysis revealed that >60% of S100-MP were highly positive for the CD54 ligand CD11a (
To corroborate that the observed effects were indeed due to an engagement of CD54 on HSC, HSC were incubated with CD54-blocking antibody or an isotype matched control antibody 2 hours prior to addition of S100-MP. CD54-blocking resulted in a significant downregulation of MMP-3 and MMP-13 induction by MP from Jurkat T cells (40% and 45%, respectively) as compared to HSC pre-incubated with the control antibody (
In order to identify (cell membrane) molecules in MP that could be implicated in the fibrolytic activation of HSC, either as ligands or as (transmembrane) signal transducing receptors, we performed proteomic analysis of S100-MP from apoptotic Jurkat T cells, with S100-MP from apoptotic Huh-7 hepatoma cells serving as negative controls. Comparative quantitative proteomics using iTRAQ isobaric tagging yielded three candidate cell-associated molecules, other than growth factor or cytokine receptors, namely Nomo-1 and Nomo-2 (molecules involved in the inhibition of TGFβ signaling, and Emmprin/Basigin (CD147) (Table 2). CD147 has been described as an inducer of MMPs, mainly MMP-1, MMP-2, MMP-3, MMP-9 and MMP-11. Of note, CD147 is activated by encounter of two CD147 positive cells, leading to homodimerization via cell-cell binding. Accordingly, FACS analysis showed that Jurkat-derived S100-MP as well as HSC were highly positive for CD147 (>70% and 99%, respectively) (
In order to define major signaling pathways that lead to MMP-induction by S100-MP, we used specific inhibitors of several kinases. MP-stimulated MMP-3 mRNA expression served as fibrolytic read-out. MMP-3 expression was completely abrogated by inhibition of p42/p44 MAP kinase (ERK1/2), while inhibition of phosphatidyl-inositol-3 (PI3) kinase/Akt did not affect MMP-3 transcript levels, and inhibition of p38 and NF-κB signaling resulted only in a modest MMP-3 mRNA suppression by 28% (
Association of MP with Other Diseases
Human Jurkat T cells (ATTC #: CRL-2570) were from ATCC (Manassas, Va.). Cells were grown in 10% fetal calf serum (FCS) in RPMI medium (with 5% CO2 in a humidified atmosphere. LX-2 human HSC were grown in 2.5% FCS in DMEM. Cells were split every 3 days at a 1:3 ratio. All media were from Cellgrow® (Manassas, Va.).
Human peripheral blood was collected in heparinized tubes from healthy volunteers within a protocol approved by the Children's Hospital, Boston, that provides anonymized blood samples. Mononuclear cells were isolated by centrifugation over Ficoll-Paque™ Premium (GE Healthcare, Uppsala, Sweden). After three washes in HBSS cells were resuspended in 10% FCS in RPMI. CD4+ and CD8+ T cells were isolated using negative selection magnetic cell sorting beads (Miltenyi Biotec, Auburn, Calif.).
Isolation of T Cell Microparticles from Plasma of Patients with Hepatitis C and Healthy Controls
Human peripheral blood was collected in citrate containing tubes from anonymized patients and healthy controls within a protocol approved by the Beth Israel Deaconess Medical Center, Boston. MP were isolated according the established protocol by differential centrifugation and the number of S100-MP was characterized by FACS w/t and with staining for Annexin V in conjunction with CD3, CD4, CD8 and CD25 (eBioscience™, San Diego, Calif.) as detailed below.
Quantification of Microparticles from Plasma of Patients with Hepatitis C, NASH, Celiac Disease, Inflammatory Bowel Disease and Healthy Controls
Human peripheral blood was collected in citrate containing tubes from anonymized patients and healthy controls within protocols approved by the Beth Israel Deaconess Medical Center, Boston. MP were isolated and standardized as to their number as above, and the relative percentage of cell specific MP determined by FACS using antibodies to CD4, CD8, CD14, CD15, CD41 (all from eBioscience™, San Diego, Calif.) and the invariant chain Valpha24/Vbeta11 (BioLegend™, San Diego, Calif. and BD Biosciences Pharmingen™, San Diego, Calif.).
Stimulation of MP Release from T Cells by Inducing Apoptosis and/or Activation
For induction of apoptosis T cells were cultured in RPMI and treated with 4 μM/mL Staurosporine (ST, Cell Signaling Technology®, Danvers, Mass.) for 4 hours. T cells were activated with 5 μg/mL Phytohemagglutinin-M (PHA, Roche, Mannheim, Germany) for 24 hours, and restimulated with PHA after 3 days. During stimulation cultures were supplemented with 5 ng/mL IL-2 (PEPROTECH®, Rocky Hill, N.J.). Three days after restimulation cells were separated from media containing MP by centrifugation at 500 g for 15 min. The cell-free supernatants were then centrifuged at 10×103 g for 20 min yielding S10-MP, while the resultant supernatant was then centrifuged at 100×103 g for 90 min to yield purified, biologically active S100-MP.
The MP preparations were characterized on a LSR2 FACS analyzer with CELLQuest software (Becton Dickinson, San Jose, Calif.). Cytometric data was further analyzed with FlowJo 7.2 (Tree Star, Inc., Ashland, Oreg.). Defined populations of particles were gated by forward and sideward scattering (FSC and SSC) acquired from runs including 500 standard beads (Becton Dickinson, San Jose, Calif.) and followed by gating for anti-CD3-APC and AnnexinV-FITC (both eBioscience™, San Diego, Calif.) double positive events. Annexin V staining of MP has previously been validated as a marker for MP. The number of double positive MP was calculated relative to the number of total beads added to the samples. The expression of CD11a and CD147 on MP was assessed using anti-CD11a- and anti-CD147-FITC (eBioscience™, San Diego, Calif.; GeneTex® Inc., Irvine, Calif., respectively).
MP membranes were labeled with the PKH26 lipid dye (Sigma-Aldrich, St. Louis, Mo.) following the manufacturer's instructions. Membrane-labeled S10- and S100-MP were coincubated with LX-2 cells for 0-1, 30 and 60 min, washed extensively and fixed with 2% paraformaldehyde for 15 min at RT. Nuclei were counterstained with the Hoechst 33342 DNA dye (Sigma-Aldrich).
HSC (200×103/well) were seeded into six-well cell culture plates (BD Labware, Franklin Lakes, N.J.) for 12 hours, serum-starved for 24 hrs, followed by incubation with 100×103 S100-MP for 1 min up to 24 hours. After incubation the HSC were washed with PBS, removed from the dishes by a short incubation with trypsin/EDTA for 5 min (0.25% Trypsin, 2.2 mM EDTA in HBSS, Cellgrow®, Manassas, Va.), and washed with FACS buffer. Single cell suspensions were stained with anti-CD3-APC in FACS buffer and CD3 receptor transfer was quantified using FACS analysis as described above.
Incubation of HSC with T Cell-Derived MP and Quantitative PCR
HSC (200×103/well) were seeded into six-well cell culture plates and serum-starved as above. HSC were then incubated with 1×103 or 50×103 S10-MP or S100-MP for 24 hours, followed by total RNA extraction from cells using TRIzol (Invitrogen, Carlsbad, Calif.). One μg of RNA was reverse-transcribed using random primers and Superscript RNase H-reverse transcriptase (Invitrogen). The sequences of primers and probes for transcripts related to fibrogenesis or fibrolysis are listed in Table 3. Target genes were mainly transcripts encoding MMPs that are capable of degrading fibrous tissue (MMP-1, 3, 9, 13) vs. COL1A1 (procollagen α1(I)) and the prominent MMP-inhibitor TIMP-1. Relative transcript levels were quantified by real-time RT-PCR on a LightCycler 1.5 instrument (Roche, Mannheim, Germany) using the TaqMan methodology as described previously (52). TaqMan probes (dual-labeled with 5′-FAM and 3′-TAMRA) and primers were designed using the Primer Express software (Perkin Elmer, Wellesley, USA), synthesized at Eurofins MWG Operon (Huntsville, Ala., USA), and validated as described by us. Experiments were performed in triplicates and values represent means±SD, being expressed as arbitrary units relative to the housekeeping gene beta2-microglobulin.
TNFα (PEPROTECH®, Rocky Hill, N.J., USA) was added to HSC cultures, and ICAM-1 expression assessed after 2, 4 and 24 hrs by flow cytometric analysis using anti-ICAM-1-FITC (eBioscience™, San Diego, Calif., USA) on a LSR2 FACS analyzer as described above.
S100-MP proteins were extracted from ST-treated Jurkat T cells and Huh-7 hepatoma cells as described above. 20 μg of membrane protein were digested with trypsin and labeled with isobaric tags (4-plex iTRAQ, Applied Biosystems, Foster City, Calif.) following the manufacturer's instructions as described, subjected to two dimensional peptide fractionation and analyzed for the comparative proteomic signature by Matrix-Assisted Laser Desorption Ionization—Time of Flight/Time of Flight Mass Spectrometry.
Subconfluent, serum-starved HSC were pre-incubated with monoclonal anti-human CD54 blocking antibody or isotype matched (IgG1) control antibody (GeneTex® Inc., Irvine, Calif., USA) at a final concentration of 50 μg/mL for 120 min, washed and incubated with Jurkat T cell derived S100-MP. S100-MP were incubated with monoclonal anti-human CD147 blocking antibody (Abcam, Cambridge, Mass., USA) or with IgG1 control antibody (GeneTex® Inc., Irvine, Calif., USA) at a final concentration of 50 μg/mL for 60 min, before being added to HSC. The effect on fibrosis-related gene expression in HSC was assessed by quantitative real-time PCR as described above.
HSC serum-starved for 24 hours were washed with ice-cold phosphate buffer, and fixed in cold methanol for 10 min. Nuclear translocation of p65 NFκB was detected by incubating cells with polyclonal p65 antibody (1:100; Delta Biolabs) for 30 min followed by TRITC-conjugated antirabbit IgG (1:200, Dako, Germany). Representative images were documented using a scanning confocal microscope (Carl Zeiss, Germany).
Pathway inhibition experiments were performed in 24 hour serum-starved HSC. The inhibitors SB203580 (p38 MAPK), U0126 (ERK1/2), and LY294002 (PI3K) (all from LC Labs, Woburn, Mass., USA) were all used at concentrations that efficiently and specifically block the respective kinase pathways in activated HSC as established previously. The proteasome inhibitor MG132 (Rockland Inc., USA) was used to block NFκB nuclear translocation and activity.
All data are given as arithmetic means with SD. Differences between values of independent experimental groups were analyzed for statistical significance by the two-tailed Student's t-test. An error level (p) <0.05 was considered significant.
24 hrs serum-starved HSC were incubated with S100-MP for 24 hrs. Apoptosis and necrosis induction by S100-MP were assessed by FACS analysis for Annexin V and 7-aminoactinomycin D staining (both from eBioscience™, San Diego, Calif., USA) on a LSR2 FACS analyzer with CELLQuest software (Becton Dickinson, San Jose, Calif.).
Primary HSCs were isolated from male Wistar rats (Retired Breeders, 450-500 g, Charles River Laboratories Int., MA, USA) according to a previously published procedure. Animal experimentation was approved by the Institutional Review Board of the Beth Israel-Deaconess Medical Center, Boston. Animals were housed with 12-hour light-dark cycles and with water and standard rat/mouse pellet chow ad libitum. Briefly, the liver was perfused with 0.1% Pronase E and 0.025% type IV collagenase in Dulbecco's modified Eagle's medium for 10-15 min, followed by digestion with 0.04% Pronase, 0.025% collagenase, and 0.002% DNase at 37° C. for 10-30 min and by a two-step centrifugation through a 11% and 13% gradient of Nycodenz at 1,500 g for 15 min. Cell viability was assessed by Trypan Blue exclusion and was routinely greater than 95-98%. Purity of HSC isolates was confirmed by their stellate shape, and cytoplasmic lipid-droplets showing greenish autofluorescence at 390 nm excitation.
Contamination with Kupffer cells, as assessed by the ability to engulf 3-μm latex beads, was 3-5% after isolation and undetectable after 10 days in first passage. Cells were used at 10 days of primary culture. Culture-activated, myofibroblast-like HSC were used between passages 3-5.
Twenty μg of membrane protein from ST-treated Jurkat T cells and Huh-7 hepatoma cells were denatured with 0.1% (v:v) SDS and then reduced by addition of 4 mM tris-(2-carboxyethyl)phosphine for one hour at 56° C. Disulfide bonds were blocked by incubation with a final concentration of 8 mM methyl methanethiosulfonate at room temperature for 10 min, followed by digestion with 10 μg Trypsin (Promega, 1 mg/ml) overnight at 37° C. Digests were labeled with the 4-plex iTRAQ isobaric tags, according to the manufacturer's protocol. Before pooling, success of labeling was confirmed by evaluating five of the highest intensity peaks on a mass spectrometer. Tagged tryptic digests were pooled and subjected to two-dimensional peptide fractionation before mass spectrometry to maximize the number of identified peptides. Pooled samples were concentrated by vacuum centrifugation and solubilized in 1 mL 10 mM KH2PO4, 25% Acetonitrile, pH 2.8, for strong cation exchange chromatography over a 4.6×100 mm POROS HS/20 column (Applied Biosystems, Foster City, Calif.) on an 1100/1200 HPLC system (Agilent Technologies, Santa Clara, Calif.) using a two-step KCl gradient at a flow rate of 0.5 mL/min over 50 minutes. Fifteen fractions were selected dried by vacuum centrifugation and resuspended in 100 μl reverse phase buffer A (2% acetonitrile, 0.1% trifluroacetic acid) and each fraction underwent reverse phase chromatography on a Dionex Ultimate NanoLC equipped with an Acclaim C18 PepMap 100 μ-precolumn followed by an analytical nanoflow C18 PepMap 100 column. Peptides were eluted with a 5%-50% gradient of acetonitrile over 60 minutes. All fractions containing peptides, based on UV absorbance at 214 nm, were directly spotted onto AB 4700 OptiTOF MALDI (Matrix-Assisted Laser Desorption Ionization) target plates using a Probot printing robot (Dionex, Sunnyvale, Calif.). Alpha-Cyano-4-hydroxycinnamic acid (CHCA) ionization matrix (Sigma-Aldrich, Saint Louis, Mo.) was mixed with the sample at a 1:2 ratio using an in-line mixing Tee in the Probot. A total of 485 fractions were collected and analyzed on the ABI 4700 MALDI-TOF/TOF MS (Matrix-Assisted Laser Desorption Ionization—Time of Flight/Time of Flight Mass Spectrometer) by tandem mass spectrometry. The 15 most abundant precursors of each spot were fragmented by MS-MS with collision-induced dissociation using medium gas pressure with ambient air. Relative abundance quantitation and peptide and protein identification were performed using GPS Explorer (Applied Biosystems, Software Revision 50861). The Swiss-Prot Homo sapiens protein database was used for all searches. The confidence value for each peptide was calculated based on agreement between the experimental and theoretical fragmentation patterns. Each protein was provided with a confidence score based on confidence scores of its constituent peptides with unique spectral patterns. Each peptide was associated with the quantitative score for each of the iTRAQ tags to calculate the relative expression levels.
Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, immunology, pharmacology, endocrinology, or related fields are intended to be within the scope of the invention.
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually incorporated by reference.
This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 61/386,232, filed Sep. 24, 2010.
This work was supported by grant number NIH 1R21DK075857-01A2 from the United States National Institutes of Health. The Government has certain rights in the invention.
Number | Date | Country | |
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61386232 | Sep 2010 | US |