Reduced Plasma Extracellular Vesicle CD5L Content in Patients With Acute-On-Chronic Liver Failure: Interplay With Specialized Pro-Resolving Lipid Mediators

Abstract

Acute-on chronic liver failure (ACLF) is a syndrome that develops in patients with acutely decompensated cirrhosis (AD). It is characterized by a systemic hyperinflammatory state, leading to multiple organ failure. Our objective was to analyze macrophage anti-inflammatory protein CD5L in plasma extracellular vesicles (EVs) and assess its as yet unknown relationship with lipid mediators in ACLF. With this aim, EVs were purified by size exclusion chromatography from the plasma of healthy subjects (HS) (n=6) and patients with compensated cirrhosis (CC) (n=6), AD (n=11) and ACLF (n=11), which were defined as positive for CD9, CD5L and CD63 and their size, number, morphology and lipid mediator content were characterized by NTA, EM, and LC-MS/MS, respectively. Additionally, plasma CD5L was quantified by ELISA in 10 HS, 20 CC and 149 AD patients (69 ACLF). Moreover, macrophage CD5L expression and the biosynthesis of specialized lipid mediators (SPMs) were characterized in vitro in primary cells. Our results indicate that circulating EVs were significantly suppressed in cirrhosis, regardless of severity, and showed considerable alterations in CD5L and lipid mediator content as the disease progressed. In AD, levels of EV CD5L correlated best with those of the SPM RvE1. Analysis of total plasma supported these data and showed that, in ACLF, low CD5L levels were associated with circulatory (p<0.001), brain (p<0.008) and respiratory (p<0.05) failure (Mann-Whitney test). Functional studies in macrophages indicated a positive feedback loop between CD5L and RvE1 biosynthesis. In summary, we have determined a significant alteration of circulating EV contents in ACLF, with a loss of anti-inflammatory and pro-resolving molecules involved in the control of acute inflammation in this condition.

Keywords: RvE1; cirrhosis; lipid autacoids; macrophages; resolution of inflammation.

Figure 1

EV characterization. Plasma fractions were pooled to generate n=6 HS, n=6 CC, n=11 AD and n=11 ACLF patient samples. (A) Upper panel: Representative graphs showing analysis of SEC eluted fractions. Left axis: Mean Fluorescence intensity (MFI) of EV markers CD9 and CD5L by bead-based flow cytometry. Right axis: protein elution was monitored by BCA. (B) CD9 MFI data of all samples analyzed. (C) Western blot of CD9 and (D) Western blot of CD63 in EVs and THP1 cell lysates, used as positive control (CT). (E) Upper panel: NTA of purified EVs from each group of patients. Lower panel: graph depicting particles/ml and size (nm) of the EV particles from 3 purifications. (F) Cryo-EM images confirmed EV presence in pooled fractions (F5-7) of SEC preparation from each group of patients. (G). CD5L and (H) the ratio of CD5L vs. CD9 MFI data. Bars represent mean ± SEM. P values were calculated using the Mann-Whitney test. HS, healthy subject; CC, Compensated Cirrhotic; AD, Acute Decompensation without ACLF; ACLF, Acute-on-Chronic Liver Failure.

Figure 2

Analysis of eicosanoid and SPM content by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics in EV from HS (n= 5), CC (n=5), AD (n=10) and ACLF (n=10) patient samples. (A) Schematic diagram of SPM biosynthesis from arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). (B) Lipid mediators are ranked on the basis of their concentration (the highest on the top; the lowest on the bottom) in the four groups. Color of bars indicates the biosynthetic pathway of the lipid mediators: green-LOX, purple-COX, and blue-Cyp450. (C, D). Total amount of lipid mediators calculated by the sum of the concentrations of each lipid mediator categorized according to the biosynthetic LOX, COX and CYP pathways (C). or to the lipid mediator biochemical family (D). Bars represent mean ± SEM. P values were calculated using the Mann-Whitney test. HS, healthy subject; CC, Compensated Cirrhotic; AD, Acute Decompensation without ACLF; ACLF, Acute-on-Chronic Liver Failure.

Figure 3

Drop of E-series resolvins in EVs from ACLF patients. Individual SPM EV content in AD vs ACLF patients. Bars represent mean ± SEM. P values were calculated using the Mann-Whitney test. Data from AD (Acute Decompensation without ACLF) (n=10) and ACLF (Acute-on-Chronic Liver Failure) (n=10) patient samples.

Figure 4

Plasma CD5L levels and RvE1 decrease in ACLF. (A) Plasma levels of CD5L were measured by ELISA in HS (n=10), CC (n=20), AD (n=80) and ACLF (n=69) patient samples (ACLF-1 n=33, ACLF-2 n=21, ACLF-3 n=15). Bars indicate the mean values ± SEM for CD5L in each group. (B) Association of CD5L with circulatory (n=17), brain (n=17) and respiratory (n=4) failures. (C) Association of CD5L with alcohol (n=63), hepatitis C virus (Hep C virus, n=37), alcohol + hepatitis C virus (Alc+virus, n=17) and other (n=18) etiologies. Data represent median plus interquartile range. (D) Spearman´s correlation heat map with correlation coefficient between CD5L and lipid mediator concentration, determined by LC-MS/MS in the plasma of AD (n=10) and ACLF (n=15) patients. (E) E series Resolvins (RvE1 and RvE2) plasma concentration, determined as in D) and in additional n=5 HS. Bars represent mean ± SEM. D. P values were calculated using the Mann-Whitney test. HS: healthy subject; CC, Compensated Cirrhotic; AD, Acute Decompensation without ACLF; ACLF, Acute-on-Chronic Liver Failure.

Figure 5

Positive feed-back between CD5L and RvE1 synthesis in macrophages. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors (n=8) by ficoll gradient centrifugation. (A) PBMCs were stimulated with 1 μg/ml CD5L or Vehicle (Veh, PBS) for 24 h, and SPMs in the supernatants were quantified by LC-MS/MS. (B–D) PBMCs were cultured for 7 days and differentiated into primary macrophages, which were stimulated with 1 μg/ml CD5L for 24 h with or without 100 ng/ml LPS during the last 4 h of culture, and the amount of mRNA encoding the indicated enzymes was quantified by RT-qPCR. (E, F) Human primary macrophages (n=5 donors) were treated for 72h with RvE1 (10 nM) with or without 10 ng/ml LPS in Vehicle (Veh, 0.007% EtOH), or 40 ng/ml Dexamethasone (DEXA), used as positive control. (E) The amount of mRNA encoding CD5L was measured by RT-qPCR. (F) Representative immunofluorescence images of CD5L (green) in primary treated Nuclei were stained with Hoechst (blue). Graphs show CD5L mean fluorescence intensity (MFI) ± SEM of 50 macrophages scored in random fields. Bars represent mean ± SEM. P values were calculated using the Mann-Whitney test.