THE IMMUNOMODULATION
EFFECTS OF ALBUMIN

PI: Joan Clària
(Hospital Clínic, Barcelona, Spain).

Promoter: EF Clif

Study 1.
In vitro studies in immune cells.

Human serum albumin (HSA) is an emerging treatment for preventing excessive systemic inflammation and organ failure(s) in patients with acutely decompensated (AD) cirrhosis. In this project, we investigated the molecular mechanisms underlying the immunomodulatory properties of HSA.

Similar to that observed during the administration of HSA to infected patients with AD cirrhosis who have elevated circulating bacterial DNA rich in CpG motifs (CpG-DNA), in isolated leukocytes HSA abolished CpG-DNA-induced cytokine expression. The reduction in cytokine levels was independent of HSA oncotic and scavenging properties. Similar anti-inflammatory effects were observed with recombinant human albumin. Moreover, HSA exerted widespread changes on the immune cell transcriptome, specifically in genes related to the endosomal compartment involved in cytosolic DNA sensing and type I interferon responses.

Using flow cytometry and confocal microscopy analyses, we demonstrated that HSA was taken up by leukocytes and internalized in vesicles positively stained with EEA1, a marker of early endosomes. Indeed, HSA and CpG-DNA colocalized in endosomes, the compartment where CpG-DNA binds to TLR9, its cognate receptor.

Furthermore, HSA also inhibited poly(I:C)- and LPS-induced IRF3 phosphorylation and TRIF-mediated responses, which are exclusive of endosomal TLR3 and TLR4 signaling, respectively. Importantly, the immunomodulatory actions of HSA did not compromise leukocyte defensive mechanisms such as phagocytosis, efferocytosis and intracellular ROS production.

Finally, the in vitro immunomodulatory effects of HSA in isolated leukocytes were confirmed in vivo in analbuminemic humanized FcRn transgenic mice.

In conclusion, these findings indicate that HSA internalizes in immune cells and modulates their responses through interaction with endosomal TLR signaling, thus providing a mechanism for the benefits of HSA infusions in patients with cirrhosis.

These studies are accepted for publication in Science Translational Journal. In addition, the intracellular pathways of HSA signaling are currently further elucidated in a collaboration with Loredana Saveanu from INSERM (Paris). In this collaboration, we explore the intracellular fate of internalized albumin and identify the endocytic pathways by which albumin is internalized using specific shRNA constructs for specific inactivation of each endocytic route.

Study 2.
Ex vivo studies in precision-cut liver and renal tissue slices.

Albumin infusions reduce the circulating levels of pro-inflammatory cytokines and improve the outcome of patients with acutely decompensated (AD) cirrhosis at risk of developing organ failure(s). In this study, we investigated whether in addition to reducing cytokine levels, albumin was able to protect tissues from the damaging actions of these inflammatory mediators. We circumscribed our investigation to TNFα, which represents a paradigmatic example of the connection between immunity and tissue injury.

Our findings indicate that incubation of hepatocytes with albumin in vitro significantly reduced TNFα-induced apoptosis by mechanisms related to inhibition of the lysosomal release of the cysteine protease cathepsin B accompanied by reductions in the cytosolic levels of pro-apoptotic signals Bid and Bax and cytochrome c released from the mitochondria, resulting in decreased caspase-3 activity. The anti-apoptotic actions of human albumin were reproduced by recombinant human albumin expressed in Oryza sativa and were confirmed ex vivo in precision-cut liver slices challenged with TNFα.

We also performed in vivo experiments in mice with pre-existing cirrhosis and expressing the human genes for albumin and neonatal Fc receptor (hAlb+/+/hFcRn+/+), which further confirmed the ability of albumin to protect against lipopolysaccharide/D-galactosamine-induced TNFalpha- mediated liver injury.

We finally translated these findings to the clinical setting and demonstrated that the hepatic apoptotic index, calculated by the plasma levels of caspase-cleaved keratin 18 to the keratin 18 ratio was significantly lower in patients with AD cirrhosis receiving human serum albumin therapy.

In conclusion, these findings uncover novel mechanisms by which albumin protects organs from cytokine-induced tissue injury, providing new avenues for understanding why albumin infusions are an effective therapy in the management of patients with AD cirrhosis.

The results of this study have been submitted to the journal Hepatology and is currently under review.

Study 3.
Translational studies based on samples obtained in interventional studies in patients with decompensated cirrhosis receiving intravenous human serum albumin (Albutein®, 20%)

We investigated the effect of albumin treatment (20% solution) on hypoalbuminemia, cardiocirculatory dysfunction, portal hypertension, and systemic inflammation in patients with decompensated cirrhosis with and without infections.

First, we assessed the effects of long-term (12 weeks) treatment with low- and high-albumin dosages (LAlbD, and HAlbD, respectively) on serum albumin, plasma renin, cardiocirculatory function, portal pressure and a large panel of cytokines in 18 non-infected patients (Pilot-PRECIOSA study). We also assessed the effect of short-term (one week) treatment with antibiotics alone versus antibiotics plus albumin on plasma cytokines in biobanking samples from 78 patients with bacterial infections included in a RCT (INFECIR-2 study).

Circulatory dysfunction and systemic inflammation were extremely unstable in many patients included in the pilot-PRECIOSA study. Long-term treatment with HAlbD but not with LAlbD was associated with normalization of serum albumin, improvement in circulation stability and left ventricular function, and suppression of keystone cytokines, without significant changes in portal pressure. The immunomodulatory effect of albumin observed in the Pilot-PRECIOSA study was confirmed in the INFECIR-2 study patients treated with albumin.

Study 4.
Immune checkpoint receptors in cirrhotic patients with acute decompensation with and without ACLF.

Checkpoint receptors are inhibitory receptors that physiologically regulate the balance between protective anti-pathogen/tumour immunity and immune-mediated tissue damage. Both chronic antigeneamia and systemic inflammation can drive hyper-expression of these receptors on immune cells, particularly T-cells, in order to switch off injurious immune responses but inadvertently promote pathogen persistence due to immune exhaustion and paralysis. We have previously shown that these receptors play a crucial role in the deranged immunity seen in Alcoholic Hepatitis (Markwick et al, Gastroenterology 2015). This dysregulated host immune response is also characteristic of patients with ACLF who present clinically with a profound immunodeficiency alongside a whole-body inflammatory response. The dynamics and expression of checkpoint receptors in ALCF is unknown.

ACLF represents an immunological paradox. Patients exhibit a hyperinflammatory state at the clinical and molecular level that exists in parallel with a profound immunoparesis and increased susceptibility to bacterial infection. We have recently demonstrated for the first time a relationship between increased gut permeability and several stimulatory and inhibitory soluble checkpoint receptors in the CANONIC cohort. Specifically, we quantified 17 soluble immune checkpoints by ELISA in the serum of 511 patients with decompensated cirrhosis included in the CANONIC Study; 334 with acute decompensation and 177 with ACLF at inclusion. We found that the soluble immune activatory checkpoints CD40 (p < 0.001), CD137 (p = 0.009) and CD27 (p = 0.003) increased significantly with increasing ACLF grade. The soluble immunosuppressive checkpoint BTLA (B and T-cell Lymphocyte-Associated) increased significantly with increasing ACLF grade (p = 0.004).

Soluble CD80, which can be both inhibitory and stimulatory, also increased with ACLF grade (p = 0.002). Galectin 9 and Galectin 1, which have numerous immunomodulatory roles, also increased with ACLF progression (p < 0.0001). Transplant-free survival at 28 days (CD40: p < 0.001, CD137: p < 0.001, CD80: p = 0.035, BTLA: p = 0.035, Galectin 9: p = 0.027, Galectin 1: p = 0.027) and 1 year (CD40: p = 0.043, CD137: p < 0.001, BTLA: p < 0.001, Galectin 9: p < 0.001, Galectin 1: p < 0.001) was also significantly associated with lower serum levels of immune checkpoints at presentation. Plotting the data as a heatmap demonstrates that levels of several checkpoint receptors increase in parallel in individual patients and this pattern is associated with increasing ACLF grade.

These findings demonstrate an association of increased levels of several stimulatory and inhibitory immune checkpoints with increasing ACLF grade. In addition, levels of certain soluble immune checkpoints can predict clinical outcome and further suggest that this activatory/inhibitory milieu is important in generating and maintaining the pathological hyperinflammatory hypoimmune state that is seen in patients with ACLF. These intriguing findings raises several mechanistic questions, which we are currently aiming to address with in vitro experiments investigating the effect of blocking these soluble checkpoint receptors on the functionality of peripheral immune cells collected longitudinally from patients with ALCF. The blockade of these receptors may reconstitute a functional immune state in ACLF and with many of these molecules being in clinical development, they may represent a tangible therapeutic opportunity for ACLF, which are severely lacking currently.

METABOLOMICS UNCOVERS A UNIQUE BLOOD METABOLITE FINGERPRINT OF PATHOPHYSIOLOGICAL RELEVANCE IN PATIENTS WITH ACLF

PI: Richard Moreau
Promoter: EF Clif

ACLF, which develops in patients with acutely decompensated (AD) cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. In this study, we aimed to analyze blood metabolome in patients with cirrhosis, with and without ACLF.

The investigation employed untargeted metabolomics using liquid chromatography coupled to high-resolution mass spectrometry in serum from 650 patients with AD (acute decompensation of cirrhosis, without ACLF), 181 with ACLF, 43 with compensated cirrhosis, and 29 healthy subjects. This investigation identified 137 annotated metabolites, of which 100 were increased in patients with ACLF of any grade, relative to those with AD, and 38 composed a distinctive blood metabolite fingerprint for ACLF. These 38 metabolites had a very close relationship with the ACLF syndrome and allow to discriminate patients with AD cirrhosis and ACLF from those with mere AD.

Moreover, in patients with ACLF, the intensity of the fingerprint increased across ACLF grades, and was similar in patients with kidney failure and in those without, indicating that the fingerprint reflected not only decreased kidney excretion but also altered cell metabolism. In addition, the fingerprint was closely associated with inflammatory markers such as tumor necrosis factor α, soluble CD206, and soluble CD163.

GENOME-WIDE ASSOCIATION ANALYSES (GWAS) IDENTIFY NOVEL GENETIC VARIANTS ASSOCIATED WITH THE ACLF SYNDROME

PI: Joan Clària
Promoter: EF Clif

We genotyped 829 patients with AD cirrhosis recruited in the CANONIC study using the Infinium® Global Screening Array (Illumina, San Diego, CA). This array has a very rich up-to-date content, includes 692,368 SNPs and is suitable for GWAS including rare variants, while also containing clinically relevant content. Prior to association analysis, a validation of the data was performed. All Caucasian patients, according to the database, were selected and the remaining patients were analysed by IBD with HAPMAP data. The genotypes with a MAF of less than a 1% were excluded from the sample. SNPs with a Hardy-Weinberg analysis p-value of less than 0.001 were also excluded. Some markers with locus missingness were also excluded. Finally, we checked the sex concordance with the database.

The final database available for GWAS analysis included 806 individuals and 500,953 SNPs. The association between each single nucleotide polymorphism (SNP) and traits relevant to the ACLF syndrome was tested using linear regression, adjusted for age, gender and ethnicity. The traits included in the analysis were ACLF, organ dysfunction or organ failure, mortality (28-day, 90-day and 6-month), markers of systemic inflammation (cytokines, CRP and WBC count), oxidative stress (HNA2, irreversible oxidized albumin) and macrophage activation (sCD206 and sCD163). The association analysis was performed using PLINK software.

Continuous variables were normalized if needed. Additional permutation tests were performed to confirm associations and to avoid false positives. The SNPs were considered significantly associated when the Bonferroni adjusted p-value was less than 5.0x10-8. In addition, we performed a replication study in 559 samples of patients with AD cirrhosis (316 samples came from (Bologna collection) and 243 samples came from (Hungary collection).

The GWAS analysis identified 17 candidate SNPs strongly associated with clinical and laboratory features of the ACLF syndrome. Two SNPs were significantly associated with the sCD206 levels (marker of macrophage activation). One SNP (rs71497223) was located within the MRC1 gene (association p-value of 1.97E-18) and was protective: it was associated with lower systemic inflammation, better prognostic scores, lower bacterial infections prevalence and lower mortality.