Palmitoylcarnitine impairs immunity in decompensated cirrhosis

Abstract

Background & aims: In patients with cirrhosis, acute decompensation (AD) correlates with a hyperinflammatory state driven by mitochondrial dysfunction, which is a significant factor in the progression toward acute-on-chronic liver failure (ACLF). Elevated circulating levels of acylcarnitine, indicative of mitochondrial dysfunction, are predictors of mortality in ACLF patients. Our hypothesis posits that acylcarnitines not only act as biomarkers, but also actively exert detrimental effects on circulating immune cells.

Methods: Plasma acylcarnitine levels were measured in 20 patients with AD cirrhosis and 10 healthy individuals. The effects of selected medium- and long-chain acylcarnitines on mitochondrial function were investigated in peripheral leucocytes from healthy donors by determining mitochondrial membrane potential (Δψm) and mitochondrial respiration using the JC-1 dye and Agilent Seahorse XF technology. Changes regarding mitochondrial ultrastructure and redox systems were assessed by transmission electron microscopy and gene and protein expression analysis.

Results: Plasma levels of several acylcarnitine species were significantly elevated in patients with AD cirrhosis compared with healthy individuals, alongside increased levels of inflammatory mediators (cytokines and chemokines). Notably, the long-chain acylcarnitine palmitoylcarnitine (C16:0-carnitine, 1.51-fold higher, p = 0.0059) impaired Δψm and reduced the spare respiratory capacity of peripheral mononuclear leucocytes. Additionally, C16:0-carnitine induced mitochondrial oxidative stress, suppressed the expression of the antioxidant gene HMOX1, and increased CXCL8 expression and IL-8 release. Etomoxir, which blocks acylcarnitine entry into the mitochondria, reversed the suppression of HMOX1. Similarly, trimetazidine, a fatty acid beta-oxidation inhibitor, prevented C16:0-carnitine-induced CXCL8 expression. Importantly, oxidative stress and Δψm impairment caused by C16:0-carnitine were less severe in the presence of albumin, a standard therapy for AD cirrhosis.

Conclusions: Our findings suggest that long-chain acylcarnitines induce mitochondrial injury in immune cells, thereby contributing to the development of immune dysfunction associated with cirrhosis.

Impact and implications: Patients with acute decompensation of cirrhosis and acute-on-chronic liver failure (ACLF) display a systemic hyperinflammatory state and leukocyte mitochondrial dysfunction. We discovered that apart from being increased in the circulation of these patients, the long-chain palmitoylcarnitine is able to elicit cytokine secretion paired with mitochondrial dysfunction in leukocytes from healthy donors. In particular, we show that inhibiting the metabolism of palmitoylcarnitine could reverse these detrimental effects. Our findings underline the importance of immunometabolism as a treatment target in patients with acute decompensation of cirrhosis and ACLF.

Keywords: Acute decompensation of cirrhosis; Acylcarnitines; Immune cells; Mitochondrial dysfunction.

Graphical abstract

Fig. 1

Levels of plasma acylcarnitines and their association with markers of inflammation. (A) Plasma levels of short-, medium- and long-chain acylcarnitines in plasma of 20 patients with acutely decompensated (AD) cirrhosis and in 10 healthy subjects (HS), displayed as median values. The Mann–Whitney U-test was used for statistical analysis. (B) Plasma levels of chemokines/cytokines in patients with AD cirrhosis in comparison with HS. (C) Correlation heatmap between acylcarnitines of different chain lengths and selected cytokines/chemokines in patients with AD cirrhosis. (D) Heatmap showing correlation between acylcarnitine and bioactive lipid mediators in patients with AD. ∗p <0.05, ∗∗p <0.01. IP-10, interferon-gamma induced protein 10; TNFα, tumour necrosis factor α.

Fig. 2

Long-chain palmitoylcarnitine reduces mitochondrial membrane potential (Δψm) and spare respiratory capacity. (A) Representative Western blot of TIM44 in PBMCs incubated with C16:0- and C6:0-carnitine. (B) Analysis of Δψm in PBMCs incubated with C16:0-carnitine by measuring the ratio of the red-to-green median fluorescence intensity emitted by the voltage-sensitive dye JC-1. (C) Mitochondrial stress test measuring the OCR in PBMCs incubated with different concentrations of C16:0-carnitine using Agilent Seahorse technology. (D) The key parameters of mitochondrial respiratory function were calculated from the OCR readings. (E) Representative electron microscopy images of PBMCs incubated for 4 h with vehicle (left panel) and C16:0-carnitine 10 μM (right panel), and analysis of mitochondrial ultrastructural parameters. Statistical differences were calculated using one-way ANOVA. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; OCR, oxygen consumption rate; PBMCs, peripheral blood mononuclear cells.

Fig. 3

Palmitoylcarnitine induces ROS production and release of pro-inflammatory cytokines. (A) Mitochondrial superoxide production in PBMCs incubated with different concentrations of C16:0-carnitines for 4 h using the indicator MitoSOX. Results are expressed as mean ± SEM. (B) Cytokines and chemokines that are significantly increased in the supernatant of PBMCs incubated with C16:0-carnitine. DMSO in vehicles 0.25% v/v. Results are expressed as median with interquartile range. Statistical differences were determined using the Kruskal–Wallis test. IFNγ, interferon-gamma; PBMCs, peripheral blood mononuclear cells; ROS, reactive oxygen species.

Fig. 4

Palmitoylcarnitine suppresses the expression of the gene coding for heme oxygenase-1 (HMOX1). (A) Volcano plot representing the pair-wise comparison of the expression level of the 84 genes related to stress and toxicity pathways in PBMCs incubated with C16:0-carnitine 10 μM for 4 h with respect to that in PBMCs incubated with control vehicle. (B) Real-time PCR confirms C16:0-carnitine as a repressor of HMOX1. (C) C16:0-carnitine suppresses heme oxygenase-1 (HO-1) protein levels in a concentration-dependent manner. (D) MnSOD mRNA expression in PBMCs incubated with increasing concentrations of C16:0-carnitine. (E) C16:0-carnitine increases the mRNA level of CXCL8 in a concentration-dependent manner. DMSO in vehicle conditions did not exceed 0.25% v/v. Results are displayed as mean ± SEM, and statistical differences were determined using the Kruskal–Wallis test. CXCL8, C-X-C Motif Chemokine Ligand 8; PBMCs, peripheral blood mononuclear cells.

Fig. 5

Etomoxir reverses the suppressive effect of palmitoylcarnitine on heme oxygenase-1. (A) Schematic diagram of the intracellular actions of etomoxir and trimetazidine. Created with BioRender.com. (B) Effect of co-treatment of C16:0-carnitine and etomoxir on HMOX1 expression. (C) Effect of co-treatment of C16:0-carnitine and etomoxir on CXCL8 expression. (D) Gene expression analysis of HMOX1 under co-treatment of C16:0-carnitine and trimetazidine. (E) Gene expression analysis of CXCL8 under co-treatment of C16:0-carnitine and trimetazidine. Results are expressed as mean ± SEM, and the Mann–Whitney U-test was applied to detect statistical differences. CPT1A, carnitine palmitoyltransferase 1A; CXCL8, C-X-C Motif Chemokine Ligand 8; HMOX1, heme oxygenase 1; TMZ, trimetazidine.

Fig. 6

Albumin restores mitochondrial membrane potential (Δψm) and abolishes ROS production elicited by palmitoylcarnitine. (A) Analysis of Δψm in PBMCs incubated with C16:0-carnitine in the presence or absence of albumin (HSA, 15 mg/ml) by measuring the ratio of the red-to-green median fluorescence intensity emitted by JC-1. (B) Mitochondrial superoxide production in PBMCs co-incubated with C16:0-carnitine and HSA. (C) HMOX1 expression in PBMCs pre-incubated with HSA and then exposed to C16:0-carnitine. (D) CXCL8 expression in PBMCs pre-incubated with HSA and then exposed to C16:0-carnitine. (E) Representative confocal images of the mitochondrial marker tom20 and HSA, with or without CCCP challenge, and quantitative analysis of co-localisation represented by Pearson’s correlation coefficient. Results are displayed as mean ± SEM, and statistical differences were detected by Kruskal–Wallis test. Scale bar: 10 μm. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CXCL8, C-X-C Motif Chemokine Ligand 8; HMOX1, heme oxygenase 1; HSA, human serum albumin; PBMCs, peripheral blood mononuclear cells; ROS, reactive oxygen species.