Liver pathophysiology

DISCOVERY: Assessing the effects of human albumin on B cells in acutely decompensated cirrhosis

The issue

The presence of organ failures in patients with acutely decompensated cirrhosis defines a syndrome called acute-on-chronic liver failure (ACLF). Patients with ACLF exhibit systemic inflammation, immunosuppression, and a high prevalence of infections leading patients to hospital and also during hospital stay (secondary infections), which are deadly complications. Although patients with ACLF exhibit neutrophilia, neutrophils from these patients have a marked reduction in the production of reactive species (ROS) stimulated by N-Formylmethionyl-leucyl-phenylalanine. Because ROS release plays a crucial role in neutrophil-mediated bacterial killing, a defect in ROS production by neutrophils may indicate immunosuppression and contribute to the elevated prevalence of infections in patients with ACLF. A recent study which used flow cytometry in blood from patients with ACLF also showed that these patients have decreases in the frequency of classical monocytes (CD14+CD16-), increases in intermediate monocytes (CD14+CD16+), and no changes in non-classical monocytes (CD14dimCD16+). Moreover, classical monocytes, but not the other monocyte subsets, had reduced expression of human leucocyte antigen DR isotype (HLA-DR). In addition, elevated frequencies of cells producing the antiinflammatory cytokine IL-10 were found among the classical and intermediate monocyte populations. Finally, transcriptional profiling of isolated classical monocytes in ACLF revealed upregulation of an array of immunosuppressive parameters and compromised antibacterial and antigen presentation machinery. Other studies mainly based on flow cytometry have shown that blood from patients with ACLF has an increased frequency of CD14+ monocytes expressing the receptor tyrosine kinase MerTK and CD14+CD15-HLADR- myeloid-derived suppressor cells. Both subsets of myeloid mononuclear cells had suppressed innate responses to bacterial PAMPs which, in addition to functional defects in neutrophils, may contribute to the elevated prevalence of infections reported in patients with ACLF. Finally, it is important to note that the lymphoid compartment in blood from patients with ACLF may also exhibit features of immunosuppression. Patients who present to hospital with ACLF have lymphopenia. Genome-wide analysis of RNA expression in blood from patients with ACLF have shown that these patients a marked reduction in RNA signatures related to CD4 T cells, CD8 T cells, natural killer cells and B cells, findings that suggest blood lymphocyte depletion and therefore immunosuppression.

To date, there are no therapies against immunosuppression in ACLF. Albumin is commonly used in patients with cirrhosis, including patients treated by large-volume paracentesis, and patients with spontaneous bacterial peritonitis, or type 1 hepatorenal syndrome. The effects of albumin were usually attributed to plasma volume expansion. Recent translational studies, however, proposed that albumin may decrease the intensity of systemic inflammation and mitigate immunosuppression in decompensated cirrhosis. Moreover, the ANSWER trial conducted in patients with decompensated cirrhosis has shown that a significant reduction in the number of episodes of bacterial infection likely contributed to the increase in the probability of survival observed in patients assigned to long-term albumin administration.

The approach

To assess if albumin mitigates immunosuppression in decompensated cirrhosis, we investigated the different populations of circulating immune cells, using their respective RNA signatures identified by whole blood RNA sequencing (RNA-seq), in 49 patients hospitalized with acutely decompensated cirrhosis who had or had not received albumin. We enrolled additional patients with acutely decompensated cirrhosis who were assigned to experiments investigating either the ex vivo effects of albumin (i.e., the effects of ex vivo exposure to albumin on bulk RNA-seq in peripheral blood mononuclear cells [PBMCs] or single cell RNA-seq [scRNA-seq] in PBMCs) or the in vivo effect of albumin assessed with the used of flow cytometry before and after albumin administration. Additional experiments also assessed the ex vivo effects of albumin on freshly isolated neutrophils.

As compared with healthy subjects, patients who had not received albumin exhibited significant changes in whole blood transcriptional profiles consistent with increases in RNA signatures of innate myeloid cells (neutrophils, monocytes, dendritic cells) and decreases in RNA signatures of lymphoid cells (T cells, B cells, and natural killer [NK] cells), findings that confirmed those showing a dichotomy in blood between the innate myeloid compartment and the lymphoid compartment. Patients receiving albumin for the usual indications (large-volume paracentesis, spontaneous bacterial peritonitis, type 1 hepatorenal syndrome) had whole blood RNA features of B cell activation associated with immunoglobulin heavy chain class switch recombination, a hallmark of differentiation of B cells into antibody-producing plasma cells. In addition, these patients had features of increased transcription related to neutrophil granules, findings consistent neutrophil activation. Of note, whole blood RNA signatures related to T cells and NK cells were not changed by albumin, findings consistent with an elective effect of albumin on RNA features related to B cells and neutrophils. In newly recruited patients with acutely decompensated cirrhosis, bulk RNA-seq in PBMCs and flow cytometry showed that albumin activated B cells, under ex vivo and in vivo conditions, respectively. Moreover, scRNA-seq of PBMCs showed that ex vivo exposure to albumin rapidly increased the RNA signatures related to transitional B cells and suggested that the action of albumin on B cells was mediated by albumin-driven interaction between regulatory intermediate monocytes and transitional B cells. Finally, additional experiments in neutrophils from patients showed that ex vivo exposure enhanced agonist-induced neutrophil degranulation and the ability of neutrophils to phagocyte and kill Candida albicans, findings consistent with albumin stimulation of neutrophil anti-microbial functions.

The objectives of this project were to determine whether the use of albumin could improve immunosuppression associated with acutely decompensated cirrhosis. Immunosuppression explains why these patients are prone to develop index or secondary infections. Treating effectively immunosuppression is an unmet medical need. Our study revealed that albumin administration was able to activate B cells and their differentiation to antibody-producing plasma cells and revigorate anti-microbial function of neutrophils. B cells and neutrophils are major players in immune responses against pathogens. Together, these findings strongly suggest that albumin mitigates, at least in part, immunosuppression by stimulating two subsets of cells involved in host immunity. The “immune-stimulating” effect of albumin may have contributed to the decrease in the number bacterial infections observed in the ANSWER study among patients who had been assigned to receive long-term albumin administration. Therefore, albumin administration may become an important component of strategies aiming to restore appropriate host immune responses to pathogens in patients with decompensated cirrhosis.