Diversity and compartmentalisation of monocytes and macrophages in patients with liver cirrhosis

Liver cirrhosis is a widespread systemic condition with increasing global prev-alence. Individuals with cirrhosis are particularly vulnerable to bacterial infec-tions, which can trigger acute decompensation (AD) and acute-on-chronic liver failure (ACLF), both of which are linked to high rates of morbidity and mortality, with limited treatment options beyond transplantation. Mononuclear phago-cytes are crucial to innate immune responses and serve as a primary defense mechanism against pathogens. The study investigated the heterogeneity of macrophages and monocytes subsets and their compartment-specific varia-tions related to disease progression in liver cirrhosis. Distinct subsets identi-fied here included AXL-expressing and CD52-expressing cells, which play critical roles in regulating innate immune responses and contribute to the complex pathogenesis of immuneparesis in patients with cirrhosis. Strong correlations with clinical parameters, cirrhosis severity scores, infectious com-plications, and mortality highlight their clinical significance, and suggest their potential as biomarkers for innate immune function integrity on the one hand and as immunotherapeutic targets on the other.
Initial investigations focused on monocyte and macrophage dysfunction in AD/ACLF stages of cirrhosis, where the risk of infection and subsequent mor-bidity and mortality is highest. Previous studies by the research group showed an increase in monocytic subsets, such as M-MDSC, occurring in compen-sated cirrhosis with increasing prevalence along with disease progression and highet proportions in AD/ACLF patients, and MERTK-expressing monocytes occurring in patients with AD/ACLF. AXL-expressing monocytes were found to increase with cirrhosis progession from mild to more severe stage, but disap-pear again in AD/ACLF stage of cirrhosis. With disease progression, these dysfunctional monocytic subsets prevailed over regular monocytes in the cir-culation of cirrhotic patients in relation to certain stages of disease. These distinct subsets exhibited unique immune-regulatory functions and immune-suppressive capabilities, compared to regular monocytes. AXL-expressing monocytes demonstrated attenuated inflammatory cytokine production and T-cell activation and enhanced efferocytosis and maintained phagocytosis of E.coli. MERTK+ monocytes have immune-regulatory function and dampen the immune response. Futhermore, those monocytes showed enhanced efferocy-tosis capacity. M-MDSC exhibit immune-suppressive properties, significantly decreasing T cell proliferation, low cytokine responses and reduced bacterial uptake.
AXL, MERTK, and Tyro-3, members of the TAM receptor tyrosine kinases (RTK) family, inhibit TLR-signaling, cytokine receptor-mediated activation, and apoptotic cell removal in monocytes/macrophages. Loss of these RTK leads to liver disease with characteristic apoptotic cell accumulation. In this study, over 90% of liver macrophages expressed AXL in homeostasis, with a reduction in AXL-expressing resident liver macrophages paralleling cirrhosis progression. Interestingly, this process was reversible, as shown by changes in AXL ex-pression in patients transitioning between Child B and Child C stages.
Immunohistochemistry indicated AXL-expressing macrophages were abun-dant in hepatic plates, while MERTK-expressing macrophages were enriched in both hepatic plates and areas of fibrosis. Phenotypically, AXL-expressing hepatic macrophages under physiological conditions were mature, indicating a need for tolerance at the portal-systemic circulation barrier. The proportion of macrophages phagocytosing gram-negative E.coli decreased in cirrhosis, with AXL-expressing monocytes showing increased migratory potential.
Compartment-specific AXL-expression was also assessed in other tissues, including the gut, peritoneum, lymph nodes, and bone marrow. Intestinal mac-rophages exhibited reduced AXL-expression with cirrhosis progression, simi-lar to hepatic macrophages. Peritoneal macrophages showed lower AXL-expression but higher MERTK-expression compared to monocyte-derived macrophages (MDM). Notably, AXL+ macrophages were absent in the bone marrow but accumulated in the circulation and lymph nodes of advanced cir-rhosis patients, supporting the hypothesis of enhanced migration potential.
These findings, which suggested a stage-specific heterogeneity in monocytes and macrophages during cirrhosis evolution, and prompted an unbiased ap-proach to investigate the underlying pathophysiology of immune. The study employed single-cell RNA sequencing (scRNA-Seq) to systematically decode the stage-specific heterogeneity of circulating monocytes in cirrhosis patients. Seven monocytic clusters were identified, representing classical, non-classical, intermediate monocytes, and M-MDSC, with varying prevalences among different cirrhosis stages. These clusters exhibited distinct marker gene expressions and functions, with inflammatory responses, phagocytosis, and complement system upregulated in compensated cirrhosis but downregulated in decompensated stages, indicating immuneparesis onset.
CD52 expression on monocytes emerged as a significant finding, with en-hanced expression in compensated and NAD cirrhosis compared to healthy controls. CD52high monocytes displayed an activated state, with increased phagocytosis capacity, cytokine production potential, and adhesion/migration behavior, and presence of CD52 expressing cells was associated with surviv-al. Interestingly, CD52 expression was downregulated or absent in AD/ACLF stages. Plasma components influenced CD52-expression levels, with bacterial particles elevating CD52 expression, while AD/ACLF plasma dampened phagocytosis capacity.
The study also explored the clinical relevance of CD52-expression on mono-cytes and soluble CD52 (sCD52) in the bloodstream as non-invasive bi-omarkers for cirrhosis severity and innate immune function. Elevated phospho-lipase C (PLC) plasma levels correlated with reduced CD52 surface expres-sion and increased sCD52 plasma levels, suggesting PLC as a potential tar-get for stabilizing CD52 on monocytes. The findings highlight the significance of the CD52 pathway as a clinically relevant biomarker and potential immuno-therapeutic target in cirrhosis management.
In conclusion, the study demonstrates that with cirrhosis progression, CD52+ and AXL+ hepatic macrophages decrease while CD52+ and AXL+ circulating monocytes increase, reflecting changes in innate immune function. The study provides insights into the potential therapeutic modulation of the AXL-GAS6 and CD52-PLC pathways to enhance innate immune responses, reduce infec-tion susceptibility, and improve survival in cirrhosis patients.