Background
Autoimmune hepatitis (AIH) is a chronic autoimmune disease of the liver, characterized by autoantibodies and elevated circulating total immunoglobulin gamma (IgG) [
1]. Diagnosis of AIH is based on a scoring system which combines, amongst others, autoantibodies, IgG level and the results of a liver biopsy [
2,
3]. However, it can still be difficult to distinguish AIH from other (autoimmune) liver diseases such as primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) or non-alcoholic fatty liver disease (NAFLD), of which prevalence is increasing. Additionally, a liver biopsy is an invasive procedure with a risk of bleeding. The addition of new, non-invasive diagnostic biomarkers in AIH would facilitate the diagnosis and might reduce the need for liver biopsy in the future.
The aim of the treatment of AIH is complete biochemical response (CBR), defined as normalization of aminotransferases and IgG. CBR is associated with a reduced disease progression and better long-term survival [
4‐
6]. While a liver biopsy is informative in disease monitoring, repeated liver biopsies are associated with high risks for complications. In clinical practice disease activity is determined by aspartate transaminase (AST), alanine transaminase (ALT) and IgG level. In some patients only partial biochemical remission can be obtained and relapses or loss of remissions can occur especially after treatment is stopped [
7]. More specific, blood-derived biomarkers would allow for a more tailored and less invasive monitoring of the patients.
The post-translational modification of proteins by
N-glycosylation adds a layer of functional complexity to the majority of human proteins, with acknowledged immunological and homeostatic roles, as differences in
N-glycan structures may influence the plasma half-life of glycoproteins as well as their functions [
8,
9]. As a major part of the plasma glycoproteins is synthesized – and glycosylated – in the liver, liver disease-induced glycosylation changes of plasma glycoproteins are of high interest. Previously, specific glycosylation patterns have been associated with non-alcoholic steatohepatitis (NASH), chronic hepatitis C virus (HCV), fibrosis, but also cirrhosis and risk of hepatocellular carcinoma (HCC) [
10‐
15]. The glycosylation pattern associated with HCC forms the basis of the clinically available GlycoCirrhoTest, specifically measuring the ratio between the proportion of bisecting
N-acetylglucosamine-containing
N-glycans and triantennary
N-glycans on circulatory glycoproteins [
10]. Distinct changes in plasma protein glycosylation have also been associated with several (autoimmune) diseases including rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and type 2 diabetes mellitus [
16‐
19].
IgG is a non-liver-derived plasma glycoprotein of which elevated levels are found in AIH [
4]. The various effector functions of IgG are highly dependent on the glycosylation (the presence and constitution of various monosaccharides) of the fragment crystallizable (Fc) region in its constant domain. For example, fucosylation of the IgG Fc glycan limits IgG-mediated antibody-dependent cellular cytotoxicity by lowering the affinity of IgG for the FcγRIII receptor [
20]. Furthermore, the degree of Fc glycan galactosylation is strongly associated with inflammation. This protein-specific glycan trait decreases with aging as well as in several (autoimmune) diseases, and dynamically relates to disease behaviour as exemplified in RA, IBD, HCV and also COVID-19 [
13,
21‐
28]. With the known association between IgG Fc galactosylation and inflammation, we hypothesize that this relationship will also be demonstrated in patients with AIH, given both the autoimmune nature and the hepatic inflammation characterizing the disease.
Studies on glycosylation in autoimmune liver disease, and AIH in particular have hitherto been lacking. Based on the promising liver disease-related plasma glycosylation effects reported in previous studies and the key role for plasma IgG levels in AIH, in the current study we aimed to extensively explore global plasma protein
N-glycosylation (the total plasma
N-glycome, TPNG) as well as IgG-specific glycosylation, in the context of differential AIH diagnosis and disease monitoring. To study glycosylation, state-of-the-art mass spectrometry (MS)-based methods were used on patient plasma material from a retrospective cross-sectional cohort [
29]. Plasma and IgG glycan levels were compared between AIH patients and healthy controls, as well as patients with PBC, PSC, NAFLD and viral or alcoholic hepatitis with different stages of cirrhosis, i.e., without cirrhosis, compensated cirrhosis or decompensated cirrhosis (WC, CC and DC, respectively). Special attention was paid to known confounders of glycosylation, including sex, age, cirrhosis, and the use of immunosuppressive medication. The primary aim of this study was to find markers (i.e., specific glycomic alterations) with diagnostic potential for AIH, reflected in the TPNG. The secondary aim was to analyse the influence of cirrhosis and disease activity on these potential novel biomarkers.
Discussion
In this cross-sectional cohort study, elevated A4GS was found as a unique marker in patients with AIH compared to healthy controls and other liver diseases. Liver inflammation and cirrhosis were shown to be important confounders influencing glycosylation patterns, but A4GS was increased in AIH patients independently of these factors. By our knowledge, this is the first study to extensively investigate glycosylation in AIH compared to healthy controls and other liver diseases and the findings offer opportunities to facilitate the non-invasive and accurate diagnosis of AIH.
Currently, diagnosis of AIH is based on the revised [
2] or the simplified [
3] AIH criteria. Although these clinical criteria exist, in practice it can be challenging to distinguish AIH from PBC, PSC and NAFLD, and a liver biopsy is in any case required for diagnosis. To reduce the invasive and risky need for liver biopsies, blood-derived markers to distinguish the diseases are desirable. We used an exploratory, MALDI-FTICR-MS-based approach for studying plasma protein
N-glycosylation, with high glycoform resolution and the ability to study sialylation and its linkages [
29]. Our approach identified high A4GS as an AIH-specific plasma
N-glycan signature, which was not only higher in AIH patients as compared to HC, but also as compared to most other liver disease groups in the cohort. This marked elevation effectively distinguished AIH patients from HC irrespective of remission status. Only patients with viral or alcoholic hepatitis without cirrhosis featured high levels of A4GS as well, but in the clinic the differentiation between viral or alcoholic hepatitis and AIH is straightforward using conventional diagnostics. The observed effect was specific for tetraantennary glycans, and independent of sialic acid linkage (Supplementary Table
5). Of note, the sialylation per galactose of di- (A2GS) and triantennary glycans (A3GS) did not show the above described trend (Supplementary Table
5), indicating it is not a global sialylation effect. Furthermore, no significant differences or associations between IgG, ALT or AST levels at diagnosis and A4GS levels were found. Despite the limited number of patients, this suggests that the high A4GS signature at diagnosis may be associated with AIH rather than the level of disease activity and may therefore be helpful in the diagnostic workup. This should be further investigated in larger studies.
Sialylated tetraantennary glycans in human plasma largely originate from alpha-1-acid glycoprotein (AGP) [
35]. AGP is a major positive acute phase protein, that functions both as an immunomodulatory as well as a transport protein, harbours 5
N-glycosylation sites and is characterized by remarkable glycosylation microheterogeneity [
37‐
39]. Hitherto, reports on AGP glycosylation mainly highlighted alterations in branching and (antennary) fucosylation to be associated with (liver) diseases [
40,
41], and more recently sialylation in NASH and HCC [
12]. The increased level of sialylation on tetraantennary glycans found in the plasma of AIH patients is a novel finding, which can either be an effect of altered regulation of sialylation or a proxy showing the upregulation of highly sialylated glycoproteins, such as AGP. Factors that regulate the levels of sialylation of circulatory proteins are, amongst others, the abundance and activity of sialyltransferases in the glycoprotein producing cells, the availability of CMP-sialic acid and the removal of non-sialylated proteins from the circulation by the asialoglycoprotein receptor (ASGPR) in the liver. While investigating the expression levels of sialyltransferases in liver cells associated with AIH would be a fruitful direction for further research, a role of the ASGPR in the observed effects seems unlikely. AIH is associated with an upregulation of ASGPR-specific autoantibodies [
42], which would have an opposite effect on the level of sialylation in the circulation, as the asialylated proteins will be limitedly removed.
While the current findings are a promising starting point in the development of a non-invasive diagnostic strategy for AIH, replication studies are required that confirm the observed effect. In addition, the marker has the potential to increase in specificity when the plasma/liver glycoproteins are identified that are responsible for the observed differences. An obvious glycoprotein candidate to further investigate is AGP and efforts to study levels and glycosylation of AGP in AIH patients are highly recommended. To consolidate our findings on the longitudinal intra-patient variation observed for A4GS and its potential to monitor disease activity over time, future prospective studies should involve longitudinal samples from treatment naïve patients with varying clinical presentations. Despite the long-term follow-up in the current study, samples were intermittently distributed in time and showed a large heterogeneity in inflammation, cirrhosis, and treatment status as well as treatment type. This, in combination with limited sample numbers, may have caused an inherent bias when longitudinal changes in altering inflammation categories were compared. Additionally, it is important to evaluate the discriminative function of the glycosylation signature in a control cohort of patients with drug-induced liver injury (DILI), since AIH and DILI are sometimes difficult to discern in clinical practice.
Other differentiators between AIH patients and HC were TPNG- and IgG bisection. The degree of cirrhosis in liver diseases vastly confounded the bisection effect in our study and this trait appeared rather cirrhosis-specific than AIH-specific, as described in literature, and exploited in the clinically approved GlycoCirrhoTest [
10,
11]. The bisection effect is likely derived from IgG, as supported by the IgG-specific data, but IgM and IgA, that are also known to carry diantennary glycans with bisecting
N-acetylglucosamines, may contribute to this observation [
35].
The level of hybrid type glycans in plasma associated with the occurrence of cirrhosis and showed a trend towards increased levels with flaring AIH. However, for the evaluation of THy as marker for disease activity, replication is needed in a more uniform set of longitudinal follow-up samples, accounting for cirrhosis and the use of medication. Higher levels of hybrid-type structures are worth further investigation in the context of AIH, as hybrid-type glycans on vitronectin have been reported to be elevated in HCC patients [
43,
44]. Conversely, plasma-derived hybrid-type glycans negatively associated with Crohn’s disease and ulcerative colitis in a cross-sectional study [
16].
Increased plasma IgG levels are one of the hallmarks of AIH, which motivated the independent investigation of IgG-specific glycosylation. Part of the galactosylation effects observed in plasma (a decreased A2G, CG, A2FG in most liver diseases) were explained by IgG-specific changes [
35]. The undergalactosylation of IgG is a known marker of ongoing inflammation, which is well documented in a broad range of diseases such as RA [
45,
46] and other autoimmune diseases [
47‐
49], IBD [
16,
22], colorectal cancer [
50], infectious diseases [
51,
52], as well as upon aging [
24‐
26,
53]. The underlying biological mechanism that might be responsible for the pro-inflammatory nature of undergalactosylated IgG is its potential ability to elicit complement activation via binding to the mannose-binding lectin [
21]. However, this causal relationship was not proven and agalactosylated total plasma IgG can also be a mere effect of the inflammatory process. In addition, complement activation does not appear to play a major role in AIH [
54]. There is a large body of evidence correlating IgG galactosylation with inflammatory markers and disease activity [
53,
55,
56], and lower IgG1 galactosylation correlated to disease activity in the current cross-sectional samples. However, no galactosylation effect was observed in the longitudinal sampling of patients changing their inflammatory status, which suggests that AIH patients in this cohort show too much heterogeneity in disease activity, cirrhosis severity and use of medication to use IgG galactosylation as marker for inflammation.
Our study focused on total plasma
N-glycosylation and IgG glycosylation in an integrated fashion, which is unique in the context of high-throughput glycomics. Still, further studies are needed to confirm the source protein from which the observed A4GS effect derives from. The currently used glycoanalytical methodologies are not yet suitable for direct clinical application. As the clinically approved GlycoCirrhoTest unfortunately does not feature sialylation analysis [
10,
11], suitable alternatives should be developed to further implement glycosylation analysis in clinical practice. These may be based on (immune)affinity assays targeting sialylation, or exploit techniques that provide a higher glycoform resolution, such as capillary electrophoresis [
57].
In conclusion, by characterizing total plasma N-glycosylation and IgG glycosylation, an AIH-specific glycosylation profile was found. High A4GS is unique for AIH and independent of remission status, which offers possibilities for development of new diagnostic markers, potentially reducing the need for liver biopsy. Secondly, it can also offer a new perspective on pathophysiology of AIH. Glycosidic changes related to disease activity should be investigated further and might aid physicians with monitoring of disease activity in the future.
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