Albumin treatment transcriptionally reprograms circulating immune cells in patients with acutely decompensated cirrhosis

BACKGROUND AND AIMS: The effects of intravenous albumin on lymphocyte perturbations and defective neutrophil anti-microbial functions that characterize patients with acute-on-chronic liver failure (ACLF) are unknown. METHODS: Forty-nine patients admitted for severe acutely decompensated cirrhosis without ACLF were investigated with the use of whole-blood RNA sequencing (RNA-seq) on admission and after a median period of 15 days once they had developed ACLF. Such patients were selected because they follow a steady systemic inflammation course. Thirty patients had received albumin during the progression to ACLF but not the 19 others. Single-cell RNA-seq (scRNA-seq) in peripheral blood mononuclear cells (PBMCs) exposed ex vivo to albumin or vehicle for 2 hours, and assessment of the anti-microbial capacity of neutrophils exposed ex vivo to albumin were performed in additional patients with acutely decompensated cirrhosis. RESULTS: Analysis of whole-blood RNA-seq data revealed that patients who had received albumin exhibited specific upregulation of signatures related to B cells, plasma cells and immunoglobulins; CD4 T cells; myeloid cells; mismatch repair, cell cycle and mitosis; and transcription factors such as c-Myc and E2F family members. The use of scRNA-seq to analyze patients' PBMCs exposed ex vivo to albumin showed increases in signatures related to B cells, myeloid cells, and CD4 T cells. Neutrophils exposed ex vivo to albumin exhibited increased chemotactic and degranulation responses, enhanced phagocytosis, and increased pathogen-destroying swarming functions. CONCLUSIONS: In patients with severe acutely decompensated cirrhosis, albumin rewires transcription in B cells, CD4 T cells and mononuclear myeloid cells, and resets neutrophil anti-microbial functions to normal. Overall design: RNA Preparation for Whole-Blood RNA-seq RNA was isolated from blood stored in Tempus tubes using the Tempus™ Spin RNA Isolation Kit (reference 43802 Applied Biosystems, Foster City, CA). Preparation for RNA-seq is detailed in the Methods section of the Supplementary Appendix. Importantly, using the HUGO Genome Nomenclature Committee, which is a resource for approved human gene nomenclature, we designed a comprehensive protocol that included not only protein-coding genes, but also genes among other gene locus types, such as immunoglobulin genes, and T-cell receptor genes, resulting in a total of 14,615 genes for analysis. Genes brought by sexual chromosomes and mitochondrial DNA were not included in the analysis. ScRNA-seq in PBMCs Peripheral venous blood (8 ml) was collected by venipuncture into EDTA-coated sterile pyrogen-free tubes (Becton Dickinson, East Rutherford, NJ) from 9 patients and 4 age-matched healthy subjects. PBMCs were immediately isolated as previously described.27 PBMCs were seeded at a density of 1.5x106 cells/ml and incubated with either albumin (Albutein®, Grifols, Barcelona, Spain) (15 mg/ml) or vehicle (culture medium) for 2 hours at 37ºC in a 5% CO2 incubator. At the end of the culture, cells were rapidly (within 30 min) transferred to ice at the Single Cell Genomics platform from the CNAG. The protocol used for scRNA-seq and analysis of data are detailed in the Methods section of the Supplementary Appendix. Experiments in Neutrophils Neutrophils were isolated from peripheral venous blood (20 ml) obtained from 6 patients and 5 age-matched healthy subjects using the Ficoll-Hypaque method or negative depletion (StemCell neutrophil purification kit). Isolated neutrophils were immediately used for different experiments that assessed major anti-microbial functions of neutrophils such as degranulation assay, phagocytosis assay, swarming, and chemotaxis. For more details, see the Methods section in the Supplementary Appendix.