Study Groups and sample collection
ACLF patients admitted to the Department of Gastroenterology, All India Institute of Medical Sciences New Delhi, were diagnosed as per the APASL (Asian Pacific Association for the Study of the Liver) criteria and recruited into this study1. The patient recruitment period was between April 2018- November 2019. The grades of ACLF were defined according to the EASL definition and categorized as grade 1, 2, and 3 depending on the number of organ systems involved. ACLF 1: patients with renal failure (creatinine ≥2.0 mg/dl) or a non-renal organ failure plus renal dysfunction (creatinine between 1.5–1.9 mg/dl) and/or HE grade I–II. ACLF 2: Patients with 2 organ failures; ACLF 3: Patients with 3 or more organ failures11. Patients with hepatocellular carcinoma or portal vein thrombosis, age group of <18 years and >75 years, diabetes (defined on the basis of recent (within 3 months) available fasting blood sugar ³126 mg/dl or random blood sugar ³200 mg/dl; were excluded from the study. During the current admission a random blood sugar ≥200 mg/dl (11.1 mmol/l) was used to define diabetes) ; presence of prior renal, respiratory and/or cardiovascular disease were excluded. Patients with impaired blood glucose levels were excluded because hyperglycaemia even without diabetes has been shown to alter basal neutrophil metabolism and cause activation12-14. ACLF patient samples were collected on the day of admission, and follow-up of 28-days was done. After 48 hours of admission, based on the blood culture reports, ACLF patients were stratified as those with sepsis or without sepsis (sterile inflammation). After the 28-day follow up period, ACLF patients were stratified as survivors and non-survivors.
Chronic liver disease (CLD) patients who were within the age group >18 years and <75 years, treatment naïve, ambulatory, without any extra-hepatic complications, and no overt symptoms were recruited from the outpatient department. Acute decompensation of CLD but without ACLF (CLD-AD) was defined as complications of cirrhosis such as jaundice, ascites, hepatic encephalopathy and GI bleed, without associated organ failure15.
Age and gender-matched healthy volunteers with no recent infection or history of past chronic illness were recruited as study controls. The study has been approved by the institute ethics committee [Reference No. IEC/473/9/2016 and, IEC/369/7/2016].
PMN were isolated from 6 ml of whole blood in EDTA within 2 hours of collection, followed by centrifugation at 500Xg for 10 minutes. Plasma was aseptically separated and stored at -80°C until further use. The remaining blood pellet used for PMN isolation as described below. Enriched PMN were used for all experiments. Overall workflow is described in Figure 1A.
PMN Isolation and Enrichment Analysis by Flow Cytometry
PMN were isolated by modified Boyum's method of double gradient centrifugation16. Blood pellet containing the buffy coat was diluted with 2X volume of sterile 1X PBS (VWR, USA, 97062-730), at room temperature (RT). Ficoll-Hisep (Himedia, INDIA, LSM 1077) was layered over Granulosep (Himedia, INDIA, LS004) in a 2:3 ratio to prepare a double gradient was prepared in a 15ml centrifuge tube. Whole blood was carefully layered on top followed by centrifugation at 300Xg for 30minutes at RT without brakes. The following phases were formed in order (top to bottom) after centrifugation: Diluted plasma, PBMC, Hisep, PMN, Granulosep, RBC pellet. Diluted plasma was discarded; PBMC layer was separated and the lower enriched PMN layer was collected. PMN cells were resuspended in sterile 1X PBS and washed twice by centrifugation at 500Xg for 10 minutes. Contaminating RBCs were removed by incubating the washed pellet in 1X RBC lysis solution for 10 minutes at 4°C, followed by two 1X PBS washes. The final cell pellet was reconstituted in 1.2 ml filtered RPMI-1640 cell culture media (Himedia, INDIA, AL171A) supplemented with 2% heat-inactivated fetal bovine serum (FBS) (Himedia, INDIA, RM10432). The enriched PMN cells were diluted 1:20 and counted by Trypan blue exclusion assay to score for live and dead cells (1µl of reconstituted cells with 10µl of 0.4% of Trypan blue and 9 µl of sterile 1X PBS). Total cells were counted, and 1x105 cells were stained with respective antibodies. To assess PMN enrichment, anti-human CD14 (clone M5E2, FITC conjugated, Cat No.301803, Biolegend, USA) and anti-human CD16 (clone 3G8, APC conjugated, Cat No 302011, Biolegend, USA) were used and their respective stain index calculated by antibody dilutions. 5x104 cells were acquired in BD LSR Fortessa X-20 Flow Cytometer. Unstained controls and single-stain tubes were prepared for each stained sample, and the acquisition was supported by BD FACSDiva software. PMN cells were gated using SSC vs FSC plot and single cells were gated as FSC height vs. FSC area (Figure 1 B-E). Neutrophils were selected as CD14- (negative) CD16+ (positive) population. The percentage enrichment of samples is listed in the Supplementary Table 1.
PMN RNA isolation and microarray
RNA isolated from 1x 106 enriched PMN cells was subjected to microarray to identify differentially expressed genes. Briefly, RNA was isolated from 1x 106 cells using the TRIzol (Invitrogen) and the manufacturer's protocol was followed. The cell suspension was pelletized and 1ml TRIzol was added to solubilize the cells by vortexing for 30 seconds. Molecular biology grade chloroform (0.2 ml) was added and mixed until milky-white appearance was formed and incubated at room temperature for 10 minutes. Phase separation was done by centrifugation at 12,700 X g for 15 minutes. The upper aqueous phase was removed carefully, and RNA was precipitated using 400 µl of ice-cold Isopropanol. The precipitate was washed in 75% ethanol before being air-dried and suspended in 30µl of RNase free water. RNA quantity and quality were checked using Bioanalyzer.
Total RNA with satisfactory integrity (RNA integrity number or RIN>7.0) and concentration, were labelled and cRNA was synthesized using Agilent Low Input Quick Amp Labelling Kit, One-Colour, without spike-in. This was followed by purification by RNeasy Mini Kit which was quantified with Nanodrop spectrophotometer (Thermofischer Scientific). The RNA yield and specific activity of Cy3 were calculated and samples meeting the manufacturer’s threshold were prepared for hybridization. 240 ng of labelled cRNA was hybridized using the Agilent Gene expression hybridization kit, onto SurePrint G3 Human gene expression v3 (Cat no. G4851C) chips, at 650C for 17 hours as per the manufacter’s protocol. The chips were washed and scanned using the Agilent surescan microarray scanner.
Gene expression Data analysis
Microarray data have been recorded and prepared according to Minimum Information About a Microarray Experiment (MIAME) guidelines and raw data have been submitted at NCBI Gene Expression Omnibus (GEO) with the accession number GSE156382. Microarray data were extracted using the Agilent Feature extraction software. The 23 individual sample feature extracted files (FEF) were converted, and data normalization was done using Genespring software followed by data analysis (Version 7.0). Briefly, the FEF files containing 56,000 probes individually, was exported to Genespring software, and sample order was chosen and grouping was done (ACLF, CLD, Healthy, ACLF sepsis, ACLF sterile inflammation). Data normalization was done using Percentile and background correction was done based on median. The normalized data was subject to sequential filtering: Expression filter of 20-100 window, Filter on flags of Detected/Non detected, Filter on Error on CV<50%, SD <0.1, SD<0.5. Statistical analysis was proceeded with the probe-set of CV <50%. NetworkAnalyst (https://www.networkanalyst.ca/NetworkAnalyst/home.xhtml) was used for carrying out DEG analysis of the mentioned sample comparatives. NetworkAnalyst uses Limma workflow for analysing data from gene expression experiments17. The differentially expressed genes were then selected with threshold criteria of p-value <0.05 and log FC >1.0 and <- 0.5 . The significant DEG were then tested for their biological pathway implication. All the gene sets from respective comparisons were taken individually for enrichment analysis. The enrichment analysis was done using ClueGO (a Cytoscape plugin) and Enrichr 18-20. The enrichment criteria for ClueGO was set for detailed network specificity at 5% genes for clustering and pathway significance of p-value < 0.05. For Enrichr, the significant pathways were selected at adjusted p-value < 0.05. KEGG enrichment analysis was used for overlapping of the DEG in both cases. Network visualization for enriched pathways. The pathways output from ClueGO was used for visualization in Cytoscape. A network centrality analysis was done for the created pathway with CytoNCA. A custom style was created wherein genes involved in various pathways were highlighted based on their expression levels and, the size of individual nodes varying to their betweenness values, which helped identify the key elements (genes/pathways) that regulate the network created. Gene set enrichment analysis (GSEA) was additionally performed using the Molecular Signatures Database or MSigDB (http://www.gsea-msigdb.org/gsea/msigdb/index.jsp).
Microarray validation and tissue-level expression by Quantitative Real-time PCR
Total PMN RNA was reverse transcribed into cDNA using verso cDNA synthesis kit (AB-1453A from Thermo Fisher Scientific). DNaseI treatment was inherent to this kit and prevented genomic DNA carryover into downstream reactions. PCR and qRT-PCR were performed with 500 ng of cDNA synthesized. from total PMN RNA. Prior to qRT-PCR, temperature gradient standardization for all primer sets were done to select optimum annealing temperature. Validation of gene expression log fold change by qRT-PCR was performed for the top 8 differentially expressed genes in ACLF vs CLD using specific primers designed from IDT oligoanalyzer software. The upregulated genes chosen for validation were ELANE, MPO, CD177, OLFM4, and OLAH. The transcript for 18S rRNA was used as a reference gene. The genes ELANE, MPO and CD177 were selected on the basis of their reported specificity in neutrophils, since the neutrophil population is known to be expanded in ACLF, resulting in a high NLR21-24.
The primer sequence and qRT-PCR conditions are included as supplementary data as per Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (See Supplementary data_MIQE guidelines file). All graphical representations of log fold change gene expression were done using Graphpad Prism.
Cell-surface CD177 Staining and Detection by Flow Cytometry
Peripheral blood was collected in EDTA vial, and 200 μl of blood was aliquoted for antibody staining. Briefly, 1ml of RBC lysis buffer was added to 200 μl of whole blood, and mixed properly. A 10X red blood cell lysis buffer was prepared in-house using NH4Cl (0.155M), KHCO3 (0.01M) and EDTA (0.1mM). The tubes were incubated for 10 minutes at 4°C, and centrifuged at 300 x g for 10 minutes. The cell pellets were washed twice with 1X PBS and suspended in 300 ul of PBS to obtain a single cell suspension. For cell surface staining, 100 μl of cell suspension was used, and the antibodies CD16 (1:100) (clone 3G8, APC conjugated, Cat No 302011, Biolegend, USA), CD66b (1:100) (clone G10F5, FITC conjugated, Cat No 305103, Biolegend, USA) and CD177 (1.5:100) (clone MEM-166, APC/Cyanine 7 conjugated, Cat No 315809, Biolegend, USA) was used. Unstained controls, stained samples, and fluorescence minus one controls were acquired on BD-LSR Fortessa flow cytometry machine. Using the FACS DIVA software, PMN gating was done based on FSC-A v/s SSC-A plot. Enriched and activated neutrophils were gated based on CD16+ CD66b+ (double positive) in a quadrant plot. CD177+ neutrophils were gated within these double positive cells.
Dual colour Immunohistochemistry for CD177 and CD16 in Post-Mortem Liver Biopsy
5-micron thick sections of formalin-fixed paraffin-embedded (FFPE) tissues were retrieved from the Department of Pathology, AIIMS New Delhi and taken on coated slides. Deparaffinization was done by dipping the slides in xylene for 5 mins (2 changes), acetone for 2-3 mins, alcohol for 2-3 mins, and then under running/tap water. Antigen retrieval was performed with citrate buffer (pH=6) in a microwave oven, at 100 degrees Celsius at 900 MW for 30 mins. Tissue sections were then allowed to cool down to come to room temperature. The slides were washed three times with Tris buffer (pH 7.5). Endogenous peroxidase blocking was done with 4% Hydrogen peroxide in 96 ml of methanol for 20 min. Anti-CD177 (Invitrogen, pH 9, 1: 50), Rabbit anti-human antibody was incubated overnight at 2-4 degrees Celsius. Next day, three sequences of washings were given with Tris buffer (pH 7.5). Universal polymer-based secondary antibody (SkyTek Laboratories, USA) was incubated at room temperature for 30 minutes, and the reaction product was developed with 3, 3"-diaminobenzidine chromogen (1: 1). Appropriate positive and negative controls were used. Colour development was monitored under the microscope. Four subsequent sequences of washings with TRIS buffer were given at 5 minutes intervals, followed by the addition of 200 microliters of enhancer and incubation for 5 minutes at room temp. After that the Mouse anti-CD16 antibody (Invitrogen, USA. 1: 50) was incubated at room temperature for 1 hr. Three sequences of washings were given with TRIS buffer. Alkaline phosphatase tagged goat anti-mouse IgG H&L secondary antibody (Ab7069) was added in a dilution of 1:10 for 30 mins at room temperature. Three sequences of washings were given in Tris buffer. A VECTOR® Blue Alkaline Phosphatase chromogen was used to develop the colour of the reaction (Blue AP), prepared in Tris HCL, pH 8.5, with 5 minutes incubation at room temperature and monitoring under the microscope. The slides were then washed under running tap water for 3-5 mins, counterstained with Neutral red before mounting with a glycerin solution. The dual-colour stained slides were photographed by using a BX43 Olympus microscope. The images taken at 2x objective power were divided into multiple fields of vision (FOVs) having a diameter of 2mm each. The procedure for manual cell counting the biopsy core was sequentially divided into multiple non-overlapping FOVs. A systematic eyeballing and counting of the cells were performed and noted. This was done to adjust the variable number of FOV available for each case due to varying biopsy core lengths. In the case of clustering and overlap of the stained cells, only those cells were counted whose nuclei were identified. Additionally, in each case, randomly in 5 FOVs the manual counting was crosschecked by the manual tagging and counting tool of `the Image Proplus 6.1 software.
All statistical analyses and graphical representations were done using Graphpad Prism software. Normally distributed continuous variables were expressed as Mean ± SD, and continuous variables with skewed distribution were expressed as Median (Interquartile Range). For two group comparisons, Unpaired T-test and Mann-Whitney Test were applied for categorical variables and non-normally distributed variables, respectively.