Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2

Abstract

Liver disease is one of the leading causes of death worldwide. Patients with cirrhosis display an increased predisposition to and mortality from infection due to multimodal defects in the innate immune system; however, the causative mechanism has remained elusive. We present evidence that the cyclooxygenase (COX)-derived eicosanoid prostaglandin E2 (PGE2) drives cirrhosis-associated immunosuppression. We observed elevated circulating concentrations (more than seven times as high as in healthy volunteers) of PGE2 in patients with acute decompensation of cirrhosis. Plasma from these and patients with end-stage liver disease (ESLD) suppressed macrophage proinflammatory cytokine secretion and bacterial killing in vitro in a PGE2-dependent manner via the prostanoid type E receptor-2 (EP2), effects not seen with plasma from patients with stable cirrhosis (Child-Pugh score grade A). Albumin, which reduces PGE2 bioavailability, was decreased in the serum of patients with acute decompensation or ESLD (<30 mg/dl) and appears to have a role in modulating PGE2-mediated immune dysfunction. In vivo administration of human albumin solution to these patients significantly improved the plasma-induced impairment of macrophage proinflammatory cytokine production in vitro. Two mouse models of liver injury (bile duct ligation and carbon tetrachloride) also exhibited elevated PGE2, reduced circulating albumin concentrations and EP2-mediated immunosuppression. Treatment with COX inhibitors or albumin restored immune competence and survival following infection with group B Streptococcus. Taken together, human albumin solution infusions may be used to reduce circulating PGE2 levels, attenuating immune suppression and reducing the risk of infection in patients with acutely decompensated cirrhosis or ESLD.

Figure 1. Elevated immunosuppressive PGE₂ in the plasma of patients admitted to hospital with Acute Decompensation (AD).

(A) LC/ESI-MS/MS was used to identify, PGE_2, among other lipids mediators 1, as being elevated in the plasma of AD patients (see supplementary Figure, n=8 for healthy volunteers and n=14 for AD patients, 3 technical replicates performed) at levels that are immunosuppressive as determined by inhibition of (B) TNFα synthesis from LPS-stimulate human monocyte-derived macrophages, (experiments were carried out on cells from n=7 healthy volunteers in duplicate). (C) Plasma from AD patients inhibited TNFα release from LPS-stimulated (200ng/ml) human monocyte-derived macrophages as well as (D) bacterial killing by these cells in a PGE₂ receptor-dependent manner (AH6809 [EP1-3 antagonist], 50μM); plasma from n=35 patients was used with experiments carried out in duplicate). The immune-suppressive effect of AD persisted for at least six days post hospitalization as determined by its EP-receptor dependent suppression of (E) TNF and elevation of (F) anti-inflammatory IL-10 using plasma from (n= 3-15 patients. Data is represented as average ± SEM. * P<0.05 ** P<0.01 ***P<0.001, ANOVA.

Figure 2. PGE₂ is immunosuppressive in patients with End Stage Liver Disease awaiting liver transplantation, but not in stable cirrhosis or non-cirrhotic liver disease.

(A) Plasma from n=13 patients awaiting liver transplant taken over 24 weeks significantly impaired LPS-stimulated macrophage TNFα synthesis in an EP receptor-dependent manner using AH6809 (EP1-3 antagonist, 50μM), with no such immunosuppression observed in plasma from (B) n=16 stable cirrhosis (Child score A) or non-cirrhotic liver disease outpatients n=5. (C) rtPCR revealed significantly elevated COX-2 (two-tail t-test, p<0.00001), but not COX-1 in peripheral blood mononuclear cells isolated from AD patients compared to healthy volunteers (n=5 per group). Both (D) bile duct ligation (BDL) and carbon tetrachloride (CCL₄) liver injury models, both revealed elevated plasma PGE₂ (n≥6 mice/group), COX 2 was found in liver Kupffer cells as well as in alveolar macrophages, micrographs from n=3 experiments are presented. * P<0.05 ** P<0.01 ***P<0.001, ANOVA.

Figure 3. Inhibiting PGE₂ reversed impaired bacterial killing and restored survival following bacterial infection.

Plasma from BDL (A) or CCL₄ (B) mice with/without the non-selective cyclooxygenase inhibitor (indomethacin, 3mg/kg p.o. to suppress circulating PGE₂ [n≥6 mice/group]) was incubated with peritoneal macrophages from naïve mice. Bile duct ligated mice were administered (C) i.p. or (D) i.v. Group B streptococcus 1h after indomethacin treatment to determine effects of inhibiting PGE₂ on bacterial killing in vivo (n≥8 mice/group) while the impact of cyclooxygenase inhibition on animal survival over time is shown in panel (E). The outcome of chronic PGE₂ inhibition on cirrhotic mouse liver is shown in (F), micrographs from n=3 experiments are presented. Data is represented as average ± SEM. * P<0.05 ** P<0.01 ***P<0.001, ANOVA.

Figure 4. PGE₂-mediated immunosuppression by AD plasma is reversed by albumin.

(A) AD plasma PGE₂ levels were corrected for corresponding albumin levels, which correlated significantly with the effects of these AD samples on LPS-stimulated human monocyte-derived macrophage TNFα synthesis. (B) AD samples were supplemented with human albumin to restore average levels back to 40 g/l (mean albumin levels in AD plasma = 25.4g/l) and then incubated with LPS-stimulated (200ng/ml) human monocyte-derived macrophages, plasma from n=19 patients were used with experiments carried out in duplicate. (C) Suppressed human monocyte-derived macrophage TNFα production caused by increasing concentrations of PGE₂ in an albumin-free culture environment is antagonized by 40mg/dl of >99% purified human serum albumin. All (D) AD plasma samples that dampened TNFα below the lowest TNFα exerted by plasma from healthy controls (Figure 1C) were arbitrarily grouped into AD^MIS (AD plasma from most immunosuppressed) with the remainder called AD^LIS (AD plasma from least immunosuppressed); albumin exerted its greatest reversal of immunosuppression on AD plasma^MIS samples. 0.5ml of 20% Human Albumin Solution (HAS) or normal saline (0.9%) was given to BDL mice (n=10/group) 1h before Group B streptococcus with (E, panel i) bacteria and (E, panel ii) PGE₂ plasma levels measured 1h later (n=5). In (F) panel (i) 20% HAS was given to AD patients (n=6) and their pre- and post (1day)-albumin plasma samples incubated with LPS-stimulated human monocyte-derived macrophages. In panel F (ii) AD plasma induced immunosuppression persisted for up to 60 days after discharge and that administration of 1L 20% HAS on days 1 & 3 had no effect on long term immunosuppression (n=10). Data is represented as average ± SEM. * P<0.05 ** P<0.01 ***P<0.001, ANOVA.