Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF

Abstract

Background and aims: Patients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients.

Material and methods: Blood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions.

Results: Several features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion.

Conclusions: Genomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies.

Keywords: adaptive immune cells; immunotherapies; innate lymphoid cells; myeloid cells; organ failure; sepsis.

Figure 1

Results of clinical blood counts and RNA microarray-inferred blood counts. (A) Clinical complete blood counts in three groups of patients with cirrhosis: AC (orange circles, n=7), AD (red square, n=7) and ACLF (purple triangle, n=17). P values were obtained using Kruskal–Wallis test and Mann–Whitney test. **P <0.01; *P <0.05. (B) Neutrophil, lymphocyte, and monocyte counts in paired clinical complete blood counts as compared with the CIBERSORT-inferred blood counts from RNA microarray data obtained with the use of peripheral blood (31 paired specimens). The shaded areas represent the 95% confidence intervals. AC denotes advanced cirrhosis, AD acute decompensation, and ACLF acute on chronic liver failure.

Figure 2

Distinctive transcriptional characteristics of ACLF. (A) Volcano plots of differential gene expression between each patients’ group [AC (n=7); AD (n=7); ACLF (n=17)] relative to healthy subjects [HS (n=7)], with significance (−log₁₀ P value) plotted against the log₂ fold-change (patients:HS ratio). Genes were considered as differentially expressed when P was <0.05 (or −log₁₀ P >1.3; dashed horizontal line) and fold-change >1.5 [or log₂ fold-change >0.58 for upregulation or log₂ fold-change <-0.58 for downregulation (right and left dashed vertical lines, respectively)]. Gray points indicate genes with no significant difference in expression, salmon indicates genes with significantly increased expression in patients and blue indicates genes with significantly decreased expression in patients. Representative differentially expressed genes are shown. CD177 was the most upregulated gene in ACLF versus HS. Between-group comparisons were performed using Student’s t-test. (B) Venn diagram showing the number of up- and downregulated genes that were unique or not to each of the three pairwise comparisons. (C) Cleveland plot showing the fold-changes for the 50 most upregulated genes in ACLF versus HS (top) and in the 50 most downregulated in this comparison (bottom). The dashed horizontal line separates upregulated genes from down regulated genes. For each gene, the fold-change of expression is also shown for two other pairwise comparisons: AD versus HS and AC versus HS. A dashed vertical line (right) indicates the threshold of 1.5 fold-change versus HS and the other dashed vertical line (left) indicates the threshold of -1.5 fold-change versus HS. (D) Results of enrichment analysis of predefined data sets (which, unless specified, were blood transcription modules (21)) with upregulated genes (left) and downregulated genes (right), both in ACLF versus HS. The enrichment of these gene sets with genes that were upregulated (left) or downregulated (right), in AC versus HS and AD versus HS genes, are also shown.

Figure 3

The CIBERSORT method identifies alterations in blood immune-cell subset composition in ACLF. There were seven healthy subjects (HS), seven patients with advanced cirrhosis (AC), seven patients with acutely decompensated cirrhosis (AD), and 17 patients with ACLF. P values were obtained using one-way ANOVA and unpaired t-tests.

Figure 4

Unique phenotype of neutrophils from patients with ACLF. (A) Volcano plots of differential neutrophil gene expression between AC and HS and ACLF versus HS. There were five healthy subjects (HS), five patients with advanced cirrhosis (AC), and five patients with ACLF. Between-group comparisons were performed using unpaired Student’s t-test. CD177 was the most upregulated gene in ACLF versus HS. (B) Enrichment of Reactome data sets with differentially expressed genes between ACLF and HS. (C) Absolute number and frequency of CD177⁺ neutrophils in blood from HS (green diamonds, n=7), patients with AC (orange circles, n=5) and patients with ACLF (purple triangles, n=7). (D) Increased adhesion to HUVECs of neutrophils from ACLF patients relative to neutrophils from HS. Neutrophils from HS (n=7) and patients (n=7) were freshly isolated; their adherence to HUVECs was measured using flow cytometry as the ratio of CD66b⁺ neutrophil per HUVEC. (E) Defective superoxide production in neutrophils from patients with ACLF is restored by a TLR7/8 agonist. Freshly isolated neutrophils from 13 patients with ACLF and 13 patients with AC were stimulated with fMLP (1 µM) and respiratory burst was monitored using the cytochrome c reduction assay. In seven patients, neutrophils were pre-treated with the TLR7/8 agonist CL097 (2 µg/ml) for 10 min before stimulation with fMLP. During the same period, median [IQR] superoxide production in neutrophils from healthy subjects was 19.6 [15.4–27.25]. ***P <0.001; *P <0.05. HUVEC denotes human umbilical vein endothelial cell, and fMLP N-Formylmethionyl-leucyl-phenylalanine.