A matched cross-sectional study of the association between circulating tissue factor activity, immune activation and advanced liver fibrosis in hepatitis C infection

Study design and population

This cross-sectional study was conducted at the Ruth M. Rothstein CORE Center in Chicago, IL between June 2011 and December 2011 and was approved by The Cook County Health and Hospitals System Institutional Review Board. Written, informed consent was obtained from each subject. Participants were stratified according to HIV and HCV status, as follows: A) HIV-monoinfection (N = 15), B) HCV-monoinfection with CHC (N = 15), C) HIV/HCV-coinfection with chronic hepatitis C (CHC) (N = 14), and D) HIV/HCV-seropositive with cleared-HCV (N = 15). Strata D consisted of both patients with spontaneous HCV clearance (N = 11) and patients who achieved a sustained virologic response following HCV therapy (N = 4). As a pilot study, sample size was determined by feasibility. Strata D was enrolled first to establish matching criteria. Subjects were matched by age, sex, estimated duration of HIV and HCV infection (HIV duration was estimated based upon self-reported year of probable high-risk exposure and if that exposure time point was unknown, then the date of diagnosis was used; HCV duration was calculated based on self-reported year of onset of high-risk activity (tattoo, exchange of sex for drugs, etc.) and if there was no clear exposure, the duration of HCV was reported as unknown), and CD4 count, with current CD4 counts in strata A and C being matched to CD4 at estimated time point of HCV clearance in strata D. All HIV-positive patients had undetectable HIV RNA. Each subject completed a questionnaire regarding HIV and HCV risk factors, had blood collected, and underwent assessment of liver fibrosis. Through the questionnaire and chart abstraction, data was collected regarding demographic factors (ethnicity), lifestyle factors (history of IDU, alcohol use, coffee consumption, smoking), and labs (D-dimer, high-sensitivity c-reactive protein (hsCRP), quantitative cytomegalovirus immunoglobulin G, complete blood count, complete metabolic panel, coagulation tests, and lipid profile) to allow for identification of potential covariates.

Liver stiffness was assessed using transient elastogroaphy (TE), a non-invasive means of assessing liver fibrosis by transmitting low speed elastic waves (~1 m/sec) and determining their propagation velocity with high speed ultrasound. All TE operators were trained and certified. Measurements were obtained via an ultrasonic probe (placed in the intercostal space at the level of the xiphoid process) perpendicular to the skin overlying the right lobe of the liver. The TE-measured liver stiffness was expressed in kilopascals (kPa) and determined from the median value of 10 “valid” measurements (ie, elastic waves that are propagated through the liver according to the machine software). The speed of wave propagation through approximately 1/500^th the volume of the liver is directly correlated with the square root of the elastic modulus or tissue stiffness [15,16]. TE-derived fibrosis scores correlate with Metavir fibrosis staging system as follows: <7.1 = stage 0–1, 7.1-9.4 = stage 2, 9.5-12.4 = stage 3, and ≥ 12.5 = stage 4. The fibrosis stage predicted by TE has been shown to correlate with the histologically determined fibrosis stage [17]. We reported liver stiffness by strata and examined its relationship with TF and immune activation markers by comparing no-moderate fibrosis (stage 0–2) to severe fibrosis (stage 3–4).

Laboratory methods

Peripheral blood was collected into heparin, EDTA, sodium citrate, and serum-separator tubes (SST) via venipuncture from patients. EDTA and sodium citrate tubes were processed within one hour. After centrifugation serum was collected from the SST tube and frozen at −80°C. Peripheral blood mononuclear cells (PBMC) were isolated from heparin tubes by density gradient centrifugation using lymphocyte separation medium (Mediatech, Inc). Cells were cryopreserved in freeze media (90% FBS + 10% DMSO) and stored in liquid nitrogen (LN₂) until testing.

Characterization of T cell immune activation and memory was performed in batch by polychromatic flow cytometry on frozen/thawed heparin PBMCs. Frozen PBMCs were removed from LN₂ storage and thawed rapidly in a 37°C water bath, washed, and rested overnight in a 37°C incubator. The following day cells were washed and stained for cell viability with Aqua Live/Dead cell stain kit (Invitrogen) prior to cell surface staining. Cell surface markers were stained with fluorochrome-conjugated monoclonal antibodies to CD3, CD8, HLA-DR, CD38, CD45RA, CCR7 (BD Biosciences), and CD4 (Invitrogen). After staining, cells were fixed in 2% formaldehyde and analyzed within 24 hours on a LSR2 flow cytometer (BD) using FACS Diva software v6.1.1. Analysis of flow cytometry data was performed using FlowJo software (Tree Star Inc). Immune activation (CD38⁺\HLA DR⁺) and memory (CD45RA\CCR7) analyses were performed after stringent gating on singlet live (Aqua⁻) CD3⁺\CD4⁺ or CD3⁺\CD8⁺ T cells.

EDTA plasma specimens were assayed in duplicate for circulating levels of IFNγ, IL-1β, IL-6, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-15, IL-17a, IP-10, MIP-1α, and TNF-α using the Human Cytokine/Chemokine kit (EMD Millipore, Billerica, MA) according to manufacturer recommended protocols. All kits were read using a Luminex 100 IS System (Luminex Corp, Austin, TX) by the Rush Proteomics and Biomarkers Core Facility with biomarker concentrations calculated using a 5-parametric curve fit using xPonent 3.2 software (Luminex Corp). Median %CV and assay recovery values all fell within acceptable limits specified by EMD-Millipore.

Plasma Preparation and MPTF Activity Measurement

Plasma specimens were collected using sodium citrate tubes and spun at 1500 × g for 15 minutes at room temperature. Platelet-poor plasma (PPP) was removed from the vacutainer tube and placed in a 15 mL conical tube and spun down a second time at 1500 × g for 15 minutes to obtain platelet-free plasma (PFP), which was transferred to a new 15 mL conical tube and vortexed to thoroughly mix. The plasma was aliquoted into 0.5 mL cryovials, flash frozen in LN₂, and stored at −80°C until analyzed.

MPTF activity was measured by a two-stage chromogenic assay as previously reported [18]. MPs were pelleted from 200 μL of PFP by centrifugation at 20 000 × g for 15 min at 4°C, washed twice with HBSA (137 mm NaCl, 5.38 mm KCl, 5.55 mm glucose, 10 mm HEPES, 0.1% bovine serum albumin, pH 7.5), and re-suspended in 100 μL of HBSA. Samples were incubated with either 1 μL of a TF-blocking antibody (hTF1) (4 μg/mL) or 1 μL of a control antibody (mouse IgG: 4 μg/mL) for 15 minutes at 25°C and then 50 μL aliquots were added to duplicate wells of a 96-well plate. Each MP sample was then incubated for 2 hours at 37°C in a 50 μL mixture of HBSA containing 10 nm factor VIIa, 300 nm factor X and 10 mm CaCl₂. Factor Xa generation was stopped by the addition of 25 μL of 25 mm EDTA buffer and 25 μL of the chromogenic substrate S2765 (4 mm) was added and incubated at 37°C for 15 min. Finally, absorbance at 405 nm was measured using a VERSAmax microplate reader (Molecular Devices). TF activity was calculated by reference to a standard curve generated using relipidated recombinant human TF (0–55 pg/mL). The TF-dependent factor Xa generation (pg/mL) was determined by subtracting the amount of factor Xa generated in the presence of hTF1 from the amount of factor Xa generated in the presence of the control antibody.

Statistical analyses

The number of patients with detectable MPTF activity versus no detectable MPTF activity (undetectable) were compared among subjects with HCV-monoinfection (strata B), those with HIV/HCV-coinfection with CHC (strata C) and those with resolved or no hepatitis C (strata A and D) using Fisher's Exact test. In addition, the quantitative detectable TF levels were compared between the strata using ANOVA. For normally distributed data, two sample t test was used to compare the difference between undetectable and detectable TF; for non-normally distributed data, Wilcoxon Rank-Sum test was used to compare the difference between undetectable and detectable TF. Mean and standard deviation for normal data, median (minimum, maximum) for non-normal data, number and percentage for categorical data were reported. P-values <0.05 were considered statistically significant. All analyses were performed using SAS Version 9.2 (Cary, NC).