Clinical management of autoimmune liver diseases: juncture, opportunities, and challenges ahead

Abstract

The three major autoimmune liver diseases are autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC).These conditions are assumed to result from a breakdown in immunological tolerance, which leads to an inflammatory process that causes liver damage.The self-attack is started by T-helper cell-mediated identification of liver autoantigens and B-cell production of autoantibodies,and it is maintained by a reduction in the number and activity of regulatory T-cells.Infections and environmental factors have been explored as triggering factors for these conditions, in addition to a genetic predisposition.Allelic mutations in the HLA locus have been linked to vulnerability, as have relationships with single nucleotide polymorphisms in non-HLA genes.Despite the advances in the management of these diseases, there is no curative treatment for these disorders, and a significant number of patients eventually progress to an end-stage liver disease requiring liver transplantation.In this line, tailored immune-therapeutics have emerged as possible treatments to control the disease.In addition, early diagnosis and treatment are pivotal for reducing the long-lasting effects of these conditions and their burden on quality of life.Herein we present a review of the etiology, clinical presentation, diagnosis, and challenges on ALDs and the feasible solutions for these complex diseases.

Introduction

Autoimmune liver diseases (ALDs) occur when our body’s immune system attacks the liver tissue, often leading to chronic inflammatory hepatic disorders, liver failure, and even cirrhosis. The most common ALDs include a) autoimmune hepatitis (AIH) clinically manifested with predominant hepatitis features [1]; b) primary biliary cholangitis (PBC, formerly named primary biliary cirrhosis) characterized by focused immune-mediated damages of the intrahepatic small bile ducts [2], and c) primary sclerosing cholangitis (PSC), a chronic cholestatic disease presented with progressive inflammatory destruction of intrahepatic and extrahepatic bile ducts [3]. Although laboratory and clinical research on their pathogenesis has significantly advanced over the past few decades, our understanding of ALD disease progression, breakthroughs in diagnosis, and treatment options for these immune-mediated diseases are much needed to do in this field [4]. Furthermore, healthcare professionals face enormous challenges in the clinical management of patients with ALD, especially those with atypical clinical manifestations for diagnosis, and respond poorly to conventional therapy (including post-transplantation treatment). Herein, we summarize the current issues, future perspectives, and challenges in diagnosing and treating patients with the three most common ALDs.

Autoimmune hepatitis (AIH)

AIH is a rare progressive chronic inflammatory liver disease with unknown etiology which affects children and adults of all ages and ethnicities [5–7]. AIH can be divided into at least two major subtypes: type1 and type2 [5, 8]. Type 1 AIH affects people of all ages, with two peaks, one in childhood/adolescence and the other in adulthood around the age of 40. Type2 AIH mainly affects children and young people [9]. Regardless of subtype, 75% of AIH patients are women [10]. Type1 AIH is associated with serological anti-nuclear antibody (ANA) with or without anti-smooth muscle antibody (SMA). Type2 AIH is associated with anti-liver kidney microsomal type 1 (anti-LKM1) antibody with or without anti-liver cytosol type 1(anti-LC1) antibody [11, 12].

AIH is usually characterized by hypergammaglobulinemia and circulating autoantibodies. Although the exact etiology is unclear, it is believed that environmental triggers and loss of immune tolerance in genetically susceptible hosts are the underlying causes of AIH. Most of these triggers are enigmatic, but some drugs and viral infections might be involved [13–16].

Pathogenesis of AIH

Genetic predisposition, molecular mimicry, immune activation, and loss of self-tolerance are considered the pathogenic mechanisms for AIH development. Genetic studies have shown that the susceptibility to AIH is partly due to polymorphisms in the human leukocyte antigen (HLA) region encoding the major histocompatibility complex (MHC). AIH is usually associated with HLA-DR3 or -DR4. In Europe and North America, type 1 AIH in adults is conferred by HLA-DR3 (HLA DRB1 * 0301) and HLA-DR4 (HLA DRB1 * 0401) genotypes [17, 18]. In Japan, Argentina, and Mexico, susceptibility is linked to HLA-DRB1*0405 and HLA-DRB1*0404 [19]. The primary susceptibility allele for pediatric type 1 AIH in Brazil and Egypt is HLA-DRB1*1301 [20, 21]. Type 2 AIH is associated with HLA-DRB1*07 and HLA-DR7-negative patients with HLA-DRB1*03 [22, 23]. Type 2 AIH is also associated with HLA-DRB1*15 (20) in Egypt. The wide heterogeneity of HLA predisposition alleles in patients with AIH indicates that differential risk factors (i.e., diet, infections, microbiome) and HLA alleles are involved in AIH development depending on the geographical locations.

Due to its location and function, the liver is constantly exposed to pathogenic antigens, malignant cells, toxins, and dietary antigens, which the hepatic immune system must be tolerant of or able to respond to [24]. Immunosuppressive cells, cytokines, and ligands in the liver offer tolerance. The hepatic sinusoids allow these cells and immunological mediators to transmigrate to the hepatic parenchyma [24]. However, the liver parenchyma is susceptible to chronic inflammation and damage when this balance is broken. T-lymphocyte-mediated cell death, an imbalance in immune cell control, and a faulty immune response to foreign antigens induced by the loss of tolerance to immune stimulants are hypothesized to constitute the pathogenesis of AIH [25].

T cell-mediated immune response plays a vital role in pathogenesis [26]. Although there are circulating autoreactive T cells in healthy individuals, their ability to cause tissue damage is limited by endogenous and exogenous peripheral tolerance mechanisms [27]. The autoimmune response in AIH is mediated by the interplay of hepatic autoantigens with various immune players. The liver is inhabited by several antigen-presenting cells (APCs), including Kupffer cells, liver sinusoidal endothelial cells (LSEC), and dendritic cells (DCs). Antigen presentation to CD4⁺ and CD8⁺ effector T cells can occur locally and affect immune tolerance [28, 29]. As depicted in Fig. 1, autoantigens can be processed by APCs in the liver and presented in the presence of MHC class II molecules to T helper 0 (Th0) cells through T cell receptor (TCR). Th0 can mature into different T helper cell subpopulations under the influence of different cytokines. In detail, Th0 can differentiate into T helper 1 cells (Th1) in the presence of interleukin (IL)−12.

Fig. 1.

Schematic diagram of AIH pathogenesis. Autoantigens are processed by APCs and presented on MHC class II molecules to Th0 cells through TCR. Exposure of autoantigens could be associated with inflammation induced by chronic infections such as HBV or HCV. These processes lead to phagocytosis and antigen presentation of cryptic antigens activating autoreactive T-cell and B-cells. Additionally, molecular mimicry between these pathogens and the human proteome may influence the loss of tolerance in the liver. In the presence of IL-12, Th0 can develop into Th1 cells, which produce IL-2 and IFN-γ. IL-2 can activate CD8.⁺ CTL to release IFN-γ and TNF and execute its cytotoxic role on hepatocytes, while IFN-γ can stimulate macrophages to release IL-1 and tumor TNF-α. Moreover, with the help of IL-4, Th0 cells can differentiate into Th2 cells, which secrete IL-4, IL-10, and IL-13. These cytokines contribute to the activation and maturation of B-cells, promoting plasma cells to secrete autoantibodies, inducing injury through complement activation and antibody-mediated cytotoxicity. Furthermore, Th0 can differentiate into Th17 cells in the presence of TGF-β, IL-1β, and IL-6. Th17 cells then produce pro-inflammatory cytokines such as IL-17, IL-22, and TNF to promote autoimmunity, including by provoking hepatocytes to secrete IL-6. Finally, the autoimmunity in AIH can be intensified by increased expression of CXCR3 on activated Th1 and Th17 and decreased numbers and Treg activity [29–41]

During inflammation, multiple foreign antigens could be immunologically crossreactive with liver tissues via molecular mimicry, leading to chronic inflammation and autoimmune mediated tissue destruction. The phenomenon of molecular mimicry was demonstrated in a animal experiment that the transfection of adenovirus carrying the primary human autoantigen CYP2D6 into mouse liver resulted in the development of AIH [30]. Furthermore, it has been shown that the hepatitis C virus (HCV) exhibits crossreactivity with liver autoantigens [30]. We note that ANA and SMA autoantibodies can be detected in approximately half of the patients with chronic hepatitis B virus (HBV) and HCV infections. About ten percent of chronic HCV patients test positive for LKM1, suggesting that molecular mimicry may play a role in the pathogenesis of AIH [31]. On the other hand, chronic inflammation and tissue destruction leading to cryptic antigen exposure can also contribute in the perpetuation of the liver immunological destruction.

All evidence mentioned above suggests that infectious and non-infectious factors are critical in breaking tolerance in the liver. This imbalance in liver immunity leads to the development of chronic inflammation in addition to Treg dysfunction. Th1 cells produce IL-2 and IFN-γ. IL-2 activates CD8⁺ cytotoxic T lymphocytes (CTL) to release IFN-γ and tumor necrosis factor (TNF) and to execute their cytotoxic role on hepatocytes expressing antigens in the context of MHC class I, and IFN-γ stimulates macrophages to release IL-1 and tumor necrosis factor-α (TNF-α) [32]. Hepatocytes exposed to IFN-γ can lead to the upregulation of MHC class I and the abnormal expression of MHC class II, resulting in further activation of T cells and perpetuation of liver injury [33, 34]. IFN-γ can also induce monocyte differentiation, promote macrophage and DC activation [42], and increase NK cell activity [35].

Th0 cells can also differentiate into T helper 2 cells (Th2) in the presence of IL-4, and Th2 can secrete IL-4, IL-10, and IL-13. These cytokines are involved in the activation and maturation of B cells, enabling plasma cells to secrete autoantibodies, inducing injury through complement activation and antibody-mediated cytotoxicity [36]. In the presence of transforming growth factor β (TGF-β), IL-1β, and IL-6, Th0 can differentiate into T helper 17 cells (Th17). Th17 can produce pro-inflammatory cytokines IL-17, IL-22, and TNF, promoting autoimmunity and inducing hepatocytes to secrete IL-6, enhancing further activation of Th17.

Regulatory T cells (Treg) are derived from Th0 cells and regulate innate and adaptive immune cells to maintain self-tolerance [37]. Intestinal microbial dysbiosis can lead to pro-inflammatory responses in the liver, causing NK cells II and CD4T lymphocytes (shows a distinct B-helper profile) to increase [38]. Further studies on the composition and functional changes of the gut microbiome in patients with AIH may shed light on the potential use of gut microbiota as non-invasive biomarkers to assess disease activity.

The specific mechanisms of the aforementioned immune cells in the pathogensis are not entirely unraveled. Studies from clinical samples and experimental animal models have demonstrated the significance of autoreactive T cells, including CD4 + T cells and CD8 + T cells infiltrating the livers of AIH patients [43]. Moreover, these CD4 + T cells and CD8 + T cells in peripheral blood correlate with disease progression [44, 45]. Renand et al [46]. found a subset of circulating autoreactive CD4 + T cells with specific autocreation to SLA and a memory PD-1 + CXCR5-CCR6-CD27 + phenotype which was only detected in AIH patients with anti-SLA autoantibodies. These specific memory PD-1 + CXCR5-CD4 + T cells were significantly increased in the blood and related to AIH disease activity [46]. You et al [47] reported that CD69 + CD103 + CD8 + tissue-resident memory T (T_RM) cells were significantly increased in the liver of patients with AIH compared to chronic hepatitis B and healthy control tissues. Moreover, CD8 + T_RM cells were correlated with AIH disease severity and significantly decreased after immunosuppression therapy [47].

The role of B cells in the pathogenesis of AIH in antigen presentation and the interaction and modulation of T-cells was examined in an experimental model of AIH. Briefly, Béland K et al [48] reported that a single dose of anti-CD20 treatment significantly reduced alanine aminotransferase levels, liver inflammation, T-cell proliferation, T follicular helper cells, together with significantly more naive and less antigen-experienced CD4 + and CD8 + T cells [48].

Activated hepatic Th17 accounts for AIH immunological injury, inflammation, and fibrosis [39]. Th17 and activated Th1 could express C-X-C motif chemokine receptor-3 (CXCR3), which binds to C-X-C motif chemokine ligands (CXCL), including CXCL9 and CXCL10. CXCL10 is considered the orchestrator of liver inflammation and fibrosis of the disease [40, 41, 49]. Th17/Treg balance is crucial in the maintenance of immune homeostasis. It has been demonstrated that the serum levels of IL-17 and IL-23 and the frequency of liver Th17 cells are increased in AIH patients when compared to other chronic hepatitis patients and healthy controls [39] whereas Treg cells are numerically and functionally impaired in AIH [50–52] when compared with that in healthy individuals [27, 53] and may contribute to the initiation of liver damage. Intriguingly, the ability of Tregs to regulate CD8 proliferation and IL-4 production can be restored after drug-induced remission, which indicates that immunosuppressive treatment can reconstitute T-regs function [54]. However, other studies have reported increasing FOXP3 + cells infiltrating the liver of AIH patients, especially during the active phase of the disease. However, these studies did not explore the regulatory function of FOXP3 + cells [55, 56].

Diagnosis of AIH

The clinical manifestations of AIH can range from asymptomatic to severe hepatitis (40, 41). Some patients have non-disease-specific symptoms, such as mild fatigue, lethargy, anorexia, nausea, pruritus, fluctuating jaundice, pain in the right upper abdomen and arthralgia, or only elevated serum aspartate transaminase (AST) and alanine transaminase (ALT), while others may already be presented with liver cirrhosis at the time of diagnosis, showing the typical signs and symptoms of chronic liver disease and some patients may be presented as acute icteric hepatitis, even liver failure [57–62].

Patients with AIH exhibit serum biochemical characteristics typical of hepatitis. The levels of bilirubin and transaminase range from normal levels to more than 50 times higher, and cholestasis enzymes are normal or slightly elevated [63, 64]. Levels of transaminases, γ-glutamyl transpeptidase (γ-GT), might also increase in AIH, which are often helpful in predicting and monitoring the response to treatment [65, 66].

In most patients, polyclonal homogammaglobulin (especially serum IgG) is elevated [5, 64, 67–69]. However, 15%−25% of patients, especially children, the elderly, and acute cases, have a normal range of IgG levels [66, 67, 69]. Therefore, the diagnosis of AIH should not be excluded just because the IgG test is normal. The diagnosis of AIH must be based on excluding all the other chronic liver diseases, such as chronic viral hepatitis, alcoholic liver disease, drug-induced hepatitis, other autoimmune liver diseases, etc.

The criteria for AIH diagnosis has developed from the report issued by the International AIH Group (IAIHG) in 1993 to the revised one in 1999 [70], and a simplified diagnostic criteria (SDC) was issued in 2008 [64] (Table 1). Liver biopsy should be considered a prerequisite for diagnosing AIH and be performed before starting treatment. In addition to diagnosis, it can be used to guide treatment decisions [34]. It should be performed in all patients with suspected AIH, including those with acute/severe or even fulminant hepatitis. A typical feature of AIH is the following:AIH is also considered likely if there is predominantly lobular hepatitis with or without centrilobular necroinflammation and at least one of the following features: portal lymphoplasmacytic hepatitis, interface hepatitis or portal-based fibrosis, in the absence of histological features suggestive of another liver disease. (Fig. 2) [71, 72].

Table 1.

Simplified diagnostic criteria for autoimmune hepatitis

Variable	Discriminator	Points
ANA or SMA	≥ 1:40	1*
ANA or SMA	≥ 1:80	2*
or LKM	≥ 1:40	2*
or SLA/LP	Positive	2*
IgG or γ- globulin level	> UNL	1
	> 1.1 times UNL	2
Liver histology	Compatible with AIH	1
(evidence of hepatitis is a necessary condition)	Typical AIH	2
	Atypical	0
Absence of viral hepatitis	Yes	2

Taken and adapted from ⁶⁴. Probable AIH ≥ 6 points; definite AIH ≥ 7 points. *Addition of points achieved for all autoantibodies (maximum, 2 points). Abbreviations: AIH autoimmune hepatitis, ANA anti-nuclear antibody, AMA antimitochondrial antibody, LKM liver kidney microsome, SMA anti-smooth muscle antibody, SLA/LP soluble liver antigen/ liver-pancreas, ULN upper limit of normal

Fig. 2.

Histological characters of the liver from autoimmune hepatitis (AIH). Interfacial hepatitis and enlarged portal area with lymphocytic/lymphoplasmacytic cell infiltration (arrow). Hematoxylin and eosin stain (HE) × 100

The US NIH Acute Liver Failure Study Group has also established autoimmune acute liver failure [57]. Like the revised SDC for AIH diagnosis [64], liver biopsy is also required to diagnose AIH. There are two different types of extensive liver necrosis. The first is severe centrilobular AIH with whole lobular necrosis, while the second is typical AIH with massive hepatic necrosis and centrilobular involvement in some cases. Other features of acute liver failure caused by AIH include portal lymphoid follicles, a plasma cell-enriched infiltrate, and central perivenulitis [57, 60, 73].

Treatment of AIH

The key objectives are ultimate remission (clinical and biochemical) of the disease and prevention of further liver disease progression. Clinical remission refers to the remission of clinical symptoms. Biochemical remission was defined as reducing transaminase and IgG levels to standard. The first-line treatment for AIH patients is corticosteroids and azathioprine (AZA) [5, 8]. Before AZA is used, the thiopurine methyltransferase (TPMT) activity of the patients should be determined, considering patients with zero or near zero TPMT activity are at risk of severe bone marrow suppression [74]. In addition, the c.415 C-to-T conversion variant of nucleoside diphosphate linked partial x motif 15 (NUDT15) gene resulted in P Arg139cys substitution is closely related to thiopurine induced bone marrow suppression and thiopurine tolerance dose, and has potential race specific genetic polymorphism [75]; The NUDT15 polymorphism was significantly associated with aza induced leukopenia in Chinese patients with AIH and related cirrhosis [76].

In 2015, the European Association for the Study of the Liver (EASL) recommended a starting dose of 0.5–1.0 mg/kg/day of prednisolone or prednisone, followed by AZA added after two weeks for treating AIH [8]. The initial dosage of AZA should be 50 mg/day and increased depending on toxicity and response up until a maintenance dose of 1–2 mg/kg/day (Table 2).

Table 2.

Treatment proposal for adult patients with AIH (e.g. 60 kg) by EASL clinical practice guidelines

Week	Prednisone (mg/d)	Azathioprine (mg/d)
1	60 (= 1 mg/kg)	-
2	50	-
3	40	50
4	30	50
5	25	100*
6	20	100*
7 + 8	15	100*
8 + 9	12.5	100*
From week 10	10	100*

Taken and adapted from⁸. Reduction of prednisolone to 7.5 mg/day if aminotransferases reach normal levels and after three-months to 5 mg/day, tapering out at three-four months intervals depending on patient’s risk factors and response. *Azathioprine (AZA) dose of 1–2 mg/kg according to body weight. Abbreviations: AIH autoimmune hepatitis, EASL European Association for the Study of the Liver

Treatment should be prolonged after reaching the biochemical endpoint to reach histological remission. Immunosuppressive treatment should be continued for at least three years and two years following complete normalization of transaminases and IgG. Though histological remission is the pathological ultimate endpoint, ensuring long-term prognosis for AIH, sustaining biochemical remission is likely the practical endpoint for AIH. Almost all patients require permanent maintenance treatment, and only a few patients can induce sustained remission after stopping all drugs. It should be noted that relapse occurs most commonly within 12 months after treatment withdrawal [77]. Therefore, patients should be closely monitored after treatment withdrawal, and surveillance should continue lifelong [78].

It is still controversial whether to perform a liver biopsy before drug withdrawal [5, 8, 67, 79, 80]. According to the 2015 EASL guidelines, to confirm histological remission, liver biopsy should be performed before drug withdrawal and at least 24 months after biochemical remission [8]. We also believe that clinical biochemical indexes should be closely monitored after discontinuation of the drug, and a follow-up biopsy should always be performed for early detection of recurrence.

Primary non-response to immunosuppressive treatment is experienced in only a small proportion of patients with AIH. For patients with ineffective first-line treatment, incomplete biochemical reaction and drug intolerance, mycophenolate mofetil (MMF) [81–83], calcineurin inhibitors, such as cyclosporine A(CsA) [84, 85] and tacrolimus (TAC) [86–90], cyclophosphamide (1–1.5 mg/kg/day) [91], methotrexate (MTX) (7.5 mg/week) [92], rituximab (1000 mg two weeks apart) [93] and infliximab [94] can be considered as second-line treatment.

Patients with severe hemocytopenia, active malignancy, pregnancy, or planned pregnancy can receive higher doses of prednisone alone (starting from 40–60 mg per day and gradually reducing to 20 mg per day within four weeks); however, a combined regimen is preferred [95]. In non-cirrhotic patients intolerant of prednisolone, budesonide is an alternative regime [8].

The 2019 AASLD practice guidance and guidelines updated their recommended first-line treatment: either prednisone monotherapy (40–60 mg/day) or a combination of prednisone (20–40 mg/day) or budesonide (9 mg/day) and AZA (50–100 mg/day) [96]. Once biochemical remission has been achieved, the dose of prednisone or prednisolone is gradually reduced to 20 mg/day or to a dose sufficient to achieve biochemical remission while monitoring laboratory tests every two weeks. Thereafter, a gradual reduction (2.5–5 mg every 2–4 weeks) is recommended to achieve a lower dose of 5–10 mg/day to maintain laboratory remission. MMF and Calcineurin inhibitors have been used to a limited extent as first-line agents in AIH. However, there is insufficient data to recommend MMF and calcineurin inhibitors as front-line agents [66, 88, 90, 96–101].

Patients with acute severe AIH should receive a trial of prednisone or prednisolone alone, while patients with acute liver failure should be directly evaluated for liver transplantation [96]. Patients with acute severe AIH without improved laboratory tests or clinically worsened within 1–2 weeks of glucocorticoid therapy should be evaluated for a liver transplant.

The incidence rate of AIH is increasing. Many patients are diagnosed with AIH after cirrhosis [102–105]. Early diagnosis and treatment of AIH vary highly between health care practices. Therefore, it is essential to develop a consistent and standardized clinical practice guideline for diagnosing and managing AIH. The choice of treatment for AIH patients who are intolerant or refractory to standard treatment is still an unresolved challenge. Moreover, the side effects of long-term steroid use also impact the quality of life. Therefore, understanding the pathogenesis of AIH is critical for effective treatment.

Experimental studies have indicated that gene mutations, contributions of gene products such as FOXP3 and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) in the pro-inflammatory/pro-B helper profile, and the infiltration of CD4 and CD8 T cells and B cells in the liver is involved in the pathogenesis of AIH [25, 38, 72, 106]. As we mentioned earlier, a reduction in Treg cells in AIH patients negatively impacts immune tolerance. Therefore, enhancing the Treg pathway is a potential therapeutic regimen to restore tolerance. Autologous T cell transfer is a potential new treatment option. However, the survival ability of transplanted Treg and the possibility of being inhibited or dedifferentiated into effector Treg cells are challenges to be resolved.

The liver homing characteristics of autologous Treg and the resulting effector T cell inhibition in AIH patients also worth further investigation [107]. Indeed, there is currently a phase I / II clinical trial evaluating the safety and efficacy of autologous T cell transplantation in AIH patients (NCT02704338). IL-2 can promote T cell proliferation, and patients with AIH have low or absent circulating levels of IL-2, leading to Treg dysfunction and self-tolerance loss [108]. A study has shown that low-dose IL-2 can promote Treg survival [109] and increase circulating Tregs [110]. Therefore, low-dose IL-2 can be used as a candidate therapy for AIH patients. There is also an ongoing phase II clinical trial to evaluate the safety and efficacy of low-dose IL-2-induced Tregs in treating various autoimmune diseases, including AIH (NCT01988506). Inhibiting the activation and proliferation of another subgroup of effector T cells is also helpful for the treatment of AIH. Current studies have shown that Sirolimus is effective in treating AIH [111, 112]. Recent studies on restoring immune tolerance by enhancing damaged regulatory T cells. It has been reported that regulating the aryl-hydrocarbon receptor signal pathway or directly enhancing CD39 expression and activity may be a novel approach for treating AIH [34, 113]. Approaches to enhance the differentiation and stimulation of Treg should be further explored.

Although AIH is mainly considered as a T cell-mediated disease, B cell-targeted therapy is also an effective method for AIH treatment. Rituximab, a CD20 monoclonal antibody that depletes B cells, effectively treats AIH [93, 114]. B-cell activating factor (BAFF) stimulates B-cell differentiation and proliferation and promotes T-cell activation [25], and serum BAFF levels are increased in patients with AIH [115]; therefore, therapies targeting BAFF might also be one of the directions of AIH treatment. Ianalumab, a human IgG1 monoclonal antibody targeting the BAFF receptor, is currently undergoing a phase II/III international randomized placebo-controlled trial for AIH therapy (NCT03217422).

Some cytokines, such as IL-1, IL-6, IL-17, and IL-23, are required for Th0 cells to differentiate into Th17 cells, and IL-12 is necessary for Th1 cell differentiation from Th0 cells [25]. Since both Th1 and Th17 cells are involved in AIH pathogenesis [31], blocking the pro-inflammatory cytokines can be a novel potential autoimmune therapy for AIH. Actually, Ustekinumab is a monoclonal immunoglobulin G1 antibody against IL-12 and IL-23, and Tocilizumab, an antagonistic antibody against the IL-6 receptor to inhibit IL-6 signaling, have demonstrated a marked clinical effect for autoimmune diseases, such as psoriatic arthritis and RA [116, 117]. Thus, it is reasonable to speculate that these recombinant monoclonal antibodies might effectively treat AIH.

Additionally, intestinal microbial disorders were involved in AIH pathogenesis by impairing intestinal barrier functions and increasing bacterial translocation.[118, 119]. Interventions to change intestinal microbiota and bacterial translocation might also be worth exploring in AIH treatment.

The latent function of bone marrow stem cells may be considered in AIH treatment. Mesenchymal stem cells (MSCs) could inhibit lymphocyte activation and proliferation and promote Treg formation, and these properties make MSCs potentially applicable in AIH treatment [120]. However, reports suggest that MSCs might promote tumorigenesis [121]. Hematopoietic stem cells (HSCs) constitute another type of stem cell with therapeutic capacity. HSCs transplantation has become a treatment option in other autoimmune diseases such as multiple sclerosis, systemic sclerosis, and rheumatoid arthritis [122]. However, studies investigating the effect of stem cells as a therapy for AIH are scarce.

Over the last few decades, there have been few clinically relevant breakthroughs in AIH treatment; corticosteroids and AZA are still considered the first-line treatment. With the gradually increased anticipation of improved quality of life and the known side effects caused by corticosteroids and AZA, it is prudent to develop new drugs and immune-based therapeutic approaches together with research directed to enhancing the understanding of the pathophysiology and immunoregulatory pathways in AIH.

Primary biliary cholangitis (PBC)

PBC, originally called "primary biliary cirrhosis", is a rare autoimmune-mediated chronic cholestatic disease. "Cirrhosis" in its previous name has been replaced by a more appropriate word, "cholangitis" in the current name because patients with this disease are increasingly diagnosed in the early stages of the disease before developing cirrhosis [123]. PBC mainly manifests as portal tract inflammation and granulomatous destruction of small intrahepatic bile ducts, predominantly affecting women. Although PBC progresses slowly, it is increasingly recognized as a disease with considerable morbidity and mortality. If it is not treated early and adequately, the prognosis of this disease would be abysmal, which might eventually lead to liver cirrhosis and liver failure [124], with a median survival time of fewer than ten years [125].

Pathogenesis of PBC

The etiology of PBC is multifactorial and still unclear. Studies suggested that the first-degree relatives of PBC patients had a higher probability of developing PBC, with 1%−2% of children of PBC patients developing the disease [126]. Among first-degree relatives (FDRs) of PBC, AMA-positive subjects have a higher risk of PBC than AMA-negative subjects (24% vs. 0.7%) [127]. AMA is a disease-specific marker in over 90% of PBC patients [128]. ANA can be found in approximately 30% of PBC patients. In AMA negative PBC patients, 5–10% develop anti-sp100 and anti-gp210 [129–132]. HLA-DR8 (DRB1*0801) was reported to be associated with increased susceptibility to PBC, with 12% of patients and 4% of controls having the risk alleles, while HLA-DRB1*11 and HLA-DRB1*13 might have protective effects [133, 134].

Data from genome-wide association studies indicated that non-HLA loci associated with antigen presentation, lymphocyte differentiation, and B cell functions are also associated with PBC [135]. Interestingly, genes located on the X chromosome may be involved in immune tolerance as some women with PBC had an increased frequency of X monosomy compared with normal people [136]. These might be accountable for the higher prevalence of PBC in women.

Epidemiological studies indicated the prevalence of PBC is higher and likely clustered in areas of low income, increased pollution, and toxic waste disposal sites [137, 138]. Smoking, history of urinary tract infection, exposure to hair dyes, and nail polish were also related to PBC [139]. In addition, infectious microorganisms such as Escherichia coli and Mycobacterium gordonae have been associated with an increased risk of PBC [126, 140–144].

The autoimmune mechanism in PBC pathogenesis is evident by the distinct B and T cell epitopes directed to a highly conserved lipoyl domain of E2 subunit of pyruvate dehydrogenase (PDC-E2), which are responsible for eliciting a cascade of immunological damage of biliary epithelial cells (BECs) of the small bile ducts[145]. Moreover, bile acid (BA), cholestasis, and the accumulation of intestinal microbiota are also involved in the pathogenesis of PBC [146].

Physiological interactions between immune and biliary pathways play significant roles in immune-mediated biliary injury and chronic cholestasis [124]. Cholestasis in PBC patients was related to impaired biliary bicarbonate secretion [147, 148]. The main bicarbonate extruder in normal human bile duct cells is the Cl^–/HCO3^–anion exchanger 2 (AE2/SLC4A2), which promotes secretin-stimulated biliary bicarbonate secretion and regulates intracellular pH [149–151]. The lack of response to secretin in PBC patients was related to the decreased expression of AE2 in bile duct cells, which might lead to cholestasis [152]. The activation of type III inositol 1,4,5-triphosphate receptor (InsP3R3) could also promote the secretion of bile bicarbonate [153]. As in AE2, the expression of InsP3R3 was down-regulated in cholangiocytes of PBC patients.

Pro-inflammatory cytokines (IL-8, IL-12, IL-17, IL-18, and TNFα) could stimulate miRNA-506 promoter activity in human cholangiocytes. miRNA-506 was overexpressed in PBC cholangiocytes and directly targeted AE2 and InsP3R3, resulting in cholestasis. Altered pH and Ca²⁺ concentrations within the BECs result in decreased mitochondrial adenosine triphosphate (ATP) production, leading to overexpression and misdirected trafficking of PDC-E2 and subsequently promoting immune activation [154]. Further, glutathione is substantially lacking during biliary cells apoptosis, and PDC-E2 can remain intact and becomes immunologically active, capable of casting a cytokine storm in the presence of AMA and macrophages in patients with PBC [155, 156]. Thus, miRNA-506 could play a vital role in the bile duct pathophysiology of PBC and likely be a potential target for disease treatment.

Additionally, it is well known that the expressions of many bile acids and drug transporters are transcriptionally inhibited by estrogen receptor alpha (ERα) activation [157]. ERα expression in the damaged small bile ducts is increased in PBC livers. A study also found that abnormal up-regulation of ERα in PBC patients positively correlated with serum pro-inflammatory cytokines (such as IL-6, IL-8, and TNFα) [158]. The pathogenesis of PBC depicted above is illustrated schematically in Fig. 3.

Fig. 3.

Schematic diagram of PBC pathogenesis. The decreased expression of AE2 and InsP3R3 in PBC leads to impaired biliary bicarbonate secretion, resulting in cholestasis in cholangiocytes. Pro-inflammatory cytokines (IL-8, IL-12, IL-17, IL-18, and TNFα) can stimulate cholangiocytes to overexpress miRNA-506, which may directly target AE2 and InsP3R3, resulting in cholestasis. Alteration of the pH and Ca²⁺ in the BECs can lead to decreased ATP production by mitochondrial, overexpression and mislocalization of PDC-E2, promoting immune activation further. Also, PDC-E2 in apoptotic bleb from bile duct cells will be recognized by AMA to intensify the autoimmunity as it remains immunologically active due to the lack of glutathione in apoptotic bodies. Furthermore, the expression of ERα in the damaged small bile ducts is increased and was positively correlated with serum pro-inflammatory cytokines (such as IL-6, IL-8, and TNFα). All those together will account for the cholestasis and the chronic damage of bile ductule in PBC [145–147, 149, 152–155, 157, 158] Abbreviation: AE2, anion exchanger 2; AMA, antimitochondrial antibody; ATP, adenosine triphosphate; BEC, biliary epithelial cell; ERα, estrogen receptor alpha; IL, Interleukin; InsP3R3, type III inositol 1,4,5-triphosphate receptor; PBC, primary biliary cholangitis; PDC-E2, E2 component of the pyruvate dehydrogenase complex; TNFα, tumor necrosis factor

Diagnosis of PBC

PBC should be highly suspected in patients with unexplained persistent cholestasis with elevated serum liver enzyme levels, and the most common being elevated alkaline phosphatase (ALP). The increased immunoglobulin concentration (particularly IgM) is another biochemical feature [159]. Serum transaminase (AST and ALT) activity may also be increased in patients, reflecting the degree of liver parenchymal inflammation and necrosis [160, 161]. AST / ALT ratio > one may be a marker of persistent liver fibrosis, and γ-GT may increase before ALP. Laboratory tests can show a decreased platelet count, low albumin concentration, and elevated international normalized ratio (INR) in patients with liver cirrhosis. PBC patients can also develop hypercholesterolemia caused by cholestasis [124]. Thus, patients with suspected PBC should have an abdominal ultrasound to exclude extrahepatic causes of cholestasis or liver tumor. MRCP imaging should be employed in those with unexplained cholestasis [124].

The guidelines focus diagnosis of PBC are based on the presence of 2 of the following three criteria [124, 162]: (1) persistent and otherwise unexplained cholestasis with an increase of ALP activity over six months; (2) positive AMA serology at a titer of more than 1:40, or positive anti-Sp100 or anti-gp210 subtypes of ANA; and (3) Histologic evidence of nonsuppurative cholangitis and interlobular bile duct injury (florid duct lesions) (Fig. 4). In most cases, liver biopsy is not necessary for diagnosis unless there is a persistent increase of ALP with PBC-specific antibodies (AMA, anti-Sp100 antibody, and anti-gp210 antibody) negative, or coexistent of AIH and PBC, or other liver diseases (steatotic liver disease) is suspected.

Fig. 4.

Histological characters of the liver from primary biliary cholangitis (PBC). The chronic inflammation is mainly limited to the portal area with lymphocytic cholangitis (arrow) but without apparent hepatocyte damage. HE × 200

PBC-related symptoms, including cholestatic pruritus, Sjogren's syndrome, abdominal discomfort, and fatigue, severely affect the quality of life [163, 164]. Typical complications (esophageal varices and bleeding, ascites, spontaneous bacterial peritonitis, and hepatic encephalopathy) in patients with advanced decompensated diseases are common. Many complications are secondary to portal hypertension and have a poor prognosis. Compared with other liver diseases, patients with PBC in pre-cirrhosis may develop portal hypertension [165].

Treatment of PBC

The EASL (European Association for the Study of the Liver) recommends that therapy in PBC should prevent end-stage complications of liver disease and managing associated symptoms [124]. Ursodeoxycholic acid (UDCA) is currently the only first-line drug approved for PBC treatment, and the international guidelines also recommend that all patients be given UDCA [124, 166]. The primary mechanism of UDCA is based on its cholagogic effect and its effect on bicarbonate secretion. UDCA can stimulate hepatocytes to secrete bile acids and promote bile secretion by activating AE2 transporter, improving the degree of cholangio-cellular injury, inflammation, and proliferation, and preventing hepatocyte injury apoptosis and necrosis, and subsequent inflammation and fibrosis. Bicarbonate can protect the bile duct epithelium from the detergent activity of hydrophobic bile salts [167, 168]. Thus, UDCA can effectively reduce ALP, bilirubin, γ-GT, cholesterol, and IgM in PBC patients. In addition, UDCA also has anti-inflammatory and immunomodulatory effects [169].

The optimal dose of UDCA is 13–15 mg/kg/day, which can be given as a single oral daily dose or a divided dose. UDCA is usually continued for a lifelong (64, 103). At present, no data indicate that UDCA has a teratogenic effect. It is considered safe to use UDCA before and during the first trimester and beyond and during breastfeeding [170, 171]. For most patients, UDCA treatment is effective. However, studies have shown that about 40% of patients develop cirrhosis after ten years [172], and approximately one-third of patients have an incomplete biochemical response to UDCA treatment [173]. Thus, there is a need for second-line therapies or combination therapies. Currently, available second-line therapies include obeticholic acid (OCA), budesonide, and fibrates.

OCA is a farnesoid-X receptor (FXR) agonist, the only drug approved by the Food and Drug Administration (FDA) in 2016 for PBC treatment after using UDCA for more than 20 years. It was recommended for the treatment of PBC in combination with UDCA with an inadequate response to UDCA only or as monotherapy for patients unable to tolerate UDCA [174], which EASL also suggested in its clinical practice guideline for PBC management [124, 166].

Though considered a second-line drug, the optimistic response rate of OCA is only around 50% (175). Dose-dependent pruritus is experienced in 56–68% of patients, leading to treatment discontinuation in ∼1–10% of patients [175]. Finally, the relatively high cost of OCA leading to substantial economic burdens on both patients and societies may limit its use. In addition, before administration of OCA in patients with advanced liver disease, it is essential to evaluate the disease, risk factors, the Child–Pugh classification, and the potential benefit from OCA before initiating this therapy. OCA would not be beneficial when a patient with liver disease classified as Child­Pugh score C or with cirrhosis classified as Child-Pugh score B. Early safety assessment should be enforced in patients with cirrhosis, and in cases of decompensation in patients taking OCA, dose adjustment or discontinuation of treatment may be required [176].

Budesonide might benefit PBC patients with 'florid' interface hepatitis on biopsy, but not in cirrhosis stage, severe osteopenia/osteoporosis, elderly patients, or patients with diabetes or hypertension. However, a phase III randomized trial of budesonide (non-cirrhotic patients) combined with UDCA (NCT00746486)[177] has been terminated after a planned interim analysis. Further scientific evidence is still needed, especially through-randomized clinical trials.

Peroxisome proliferator-activated receptors (PPAR) agonists may be a potential treatment for PBC. Fibrates target PPAR, of which there are three subtypes: α, δ, and γ [178]. PPAR α is involved in bile acid synthesis, transport, and metabolism [178, 179]. PPAR δ is involved in cholesterol transport and excretion from the biliary epithelium. PPAR γ has been shown to inhibit nuclear factor κ B activation, and loss of this inhibition might be associated with autoimmunity [180]. Bezafibrate shows similar specificity for all three isoforms. It can induce a rapid and sustained normalization of serum ALP values in PBC with the added benefit of alleviating cholestatic pruritus (NCT01654731, NCT02701166) [181, 182]. Fenofibrate acts on PPAR α with a high degree of specificity (NCT00575042) [178, 183], Seladelpar is a specific agonist of PPAR δ [184], Elafibranor is a dual agonist of PPAR α and δ [185], and Saroglitazar is a dual agonist of PPAR α and γ (NCT03112681) [186]. Currently, Seladelpar and Elafibrinor have been already approved by FDA, other drugs are currently in the animal or clinical trial stage.

Fibrates might be suitable for patients with obvious hyperlipidemia and high cardiovascular risk. Patients with pruritus as the primary symptom have potential side effects, such as muscle injury (myositis and rhabdomyolysis) and renal insufficiency. It may also cause drug-induced liver damage [187, 188]. In addition, there are some immunosuppressive and immunomodulatory drugs, including AZA [189], MTX [190], thalidomide [191], colchicine [192], cyclosporine [193], and MMF [194]. However, there was no satisfactory effect shown in these studies. Due to the low bioavailability of OCA and associated side effects such as pruritus, novel FXR agonists are being developed for the treatment of PBC, including cilofexor (NCT02943447), tropifexor (NCT02516605) [195], and EDP-305 (NCT03394924) [196].

Fibroblast growth factor (FGF) 19, the downstream target of FXR, acts on hepatocytes and inhibits CYP7A1 expression and bile acid synthesis[197]. In a phase 2 trial, NGM282 has been shown to reduce ALP in patients who did not respond to UDCA (NCT02026401) [198].

Activation of vitamin D receptors is associated with anti-inflammation, anti-cholestasis, and anti-fibrosis. The expression of Vitamin D receptor is elevated in immune cells, and its activation can reduce effector T cell responses by suppressing Th1 cell generation and promoting Th2 cell generation [168]. Therefore, vitamin D receptors may be a potential target in PBC treatment.

Due to the complexity of the immunobiology of PBC, we believe that new immunotherapeutic methods such as immunosuppressants and immunomodulators (such as anti-IL-12, anti-CD80, anti-CD20 [rituximab]) [199–202], mesenchymal stem cells [203, 204] and CTLA4 (abatacept) [205] may be effective for PBC. Some other target sites, such as fractalkine/CX3C chemokine ligand 1 (CX3CL1) [206] inhibitor, sphingosine-1-phosphate receptors (S1PR) 1 and S1RP4 agonists [207], nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) 1, and NOX4 inhibitor [208, 209], transmembrane G-protein-coupled receptor 5 agonists (TGR5) [210], apical sodium-dependent bile acid transporter (ASBT) inhibitor are also currently being explored as potential drugs for the treatment of PBC. Pruritus is a common complication of cholestatic liver disease. The pathogenesis of cholestatic pruritus is very complex. As a key element of bile acid enterohepatic circulation, ABST may be a method to treat cholestatic pruritus by reducing bile acid pool through ASBT inhibitor. Linerixibat is a minimally absorbed, selective small molecule inhibitor of the ileal bile acid transporter, IIb Phase clinical trials have shown that it can alleviate pruritus, sleep disturbance, and depression(NCT02966834) [211].

The intestinal microenvironment is considered to play a role in the pathogenesis of PBC, and intestinal microbiota translocation can promote autoimmune cholangitis [212]. The enzymes produced by the intestinal microbiota are considered to be deconjugate and dehydroxylating bile acids [213]. Therefore, regulating the intestinal microbiota may also be helpful for treatment. The current approved and potential drugs for PBC treatment are listed in Table 3.

Table 3.

Approved and potential treatment drugs of PBC

Treatment	Mechanism	Current status	NCT number	Reference
Approved treatments
UDCA	Cholagogic, promote bicarbonate secretion	First-line drug	-	124
OCA	FXR agonist	Second-line drug	-	124
Seladelpar	PPAR δ agonist	Second-line drug		184
Elafibranor	PPAR α and δ agonist	Second-line drug		185
Off-label treatments
Budesonide	Immunosuppression	Phase III randomized	NCT00746486	177
Bezafibrate	PPAR agonist	Phase III randomized, completed Phase II randomized, recruiting	NCT01654731 NCT02701166	181,182
Fenofibrate	PPAR α agonist	Phase II randomized, completed	NCT00575042	178,183
Saroglitazar	PPAR α and γ agonist	Phase II randomized, completed	NCT03112681	186
Cilofexor	FXR agonist	Phase II randomized, completed	NCT02943447	-
Tropifexor	FXR agonist	Phase II randomized, completed	NCT02516605	195
EDP-305	FXR agonist	Phase II randomized, completed	NCT03394924	196
NGM282	FGF19 agonist	Phase II randomized, completed	NCT02026401	198
Vitamin D	Vitamin D receptor agonist	Animal trial	-	168
Rituximab	Immunosuppression	Phase I & II randomized, completed	NCT00364819	199–202
MSC	Inhibit T cells proliferation	Phase II randomized, recruiting	-	120
Abatacept	Immunosuppression	Animal trial	-	205
E6011	CX3CL1 inhibitor	Phase II randomized, terminated	NCT03092765	206
Etrasimod APD334	S1PR1 and S1RP4 agonists	Phase II randomized, terminated	NCT03155932	207
GKT137831	NOX1 and NOX4 inhibitor	Phase II randomized, completed	NCT03226067	209
TGR5 agonists	TGR5 agonists	Animal trial	-	210
Linerixibat	ASBT inhibitor	Phase IIb randomized, completed	NCT02966834	211
Probiotic	Change gut microenvironment	Phase II randomized, planed	NCT03521297	212

CX3CL CX3C chemokine ligand, FGF fibroblast growth factor, FXR farnesoid-X receptor, MSCs Mesenchymal stem cells, NCT national clinical trial, NOX nicotinamide adenine dinucleotide phosphate hydrogen oxidase, OCA obeticholic acid, PPAR peroxisome proliferator-activated receptors, S1PR sphingosine-1-phosphate receptors, TGR5 transmembrane G-protein-coupled receptor 5 agonists, UDCA ursodeoxycholic acid

As UDCA will not improve pruritus, fatigue, Sjogren's syndrome, and other discomforts, Cholestyramine (first-line) and rifampicin (second-line) can be taken as therapy for pruritus [214, 215]. There is still no recommended treatment for fatigue caused by PBC. Clinicians should also actively look for possible causes, especially anemia, hypothyroidism, and sleep disorders, to relieve fatigue. The application of artificial tears and saliva may be conducive to mitigating dry symptoms. Lastly, liver transplantation is necessary when drug treatment fails to control disease progression and liver cirrhosis complications [124].

Collectively, treatments for PBC are primarily directed at symptoms than controlling autoimmunity. Drugs such as UDCA often have insufficient efficacy, especially in advanced diseases. Liver transplantation sometimes seems to be the only option. Most patients need to take life-long medication, and their symptoms such as fatigue, pruritus, dry mouth, and eye are difficult to control and relieve. All these lead to their low quality of life. Thus, effort directed toward developing novel drugs and a deeper understanding of the molecular mechanisms underlying the pathophysiology, genetic basis, and pathogenesis of PBC will cast hope on preventing the deterioration of symptoms, halting disease progression, and even curing PBC.

Primary sclerosing cholangitis (PSC)

PSC is an immune-mediated chronic progressive liver disease histologically characterized by intrahepatic and/or extrahepatic cholangitis. Eventually, it leads to intrahepatic/extrahepatic bile duct fibrosis and multifocal strictures, cholestasis, and liver cirrhosis. PSC is a relatively rare disease and is more common among young people. The incidence rate of males is higher than that of females [216–218], and the rate in northern Europe and parts of the United States is higher than that in southern Europe and Asia [218]. Although the onset of PSC is usually insidious, its natural course can lead to liver cirrhosis, hepatobiliary malignancies, colon cancer, and superimposed bacterial cholangitis [219–222]. Cholangiocarcinoma in PSC patients is about 10—12% [222–224]. The median death or liver transplantation time is 15–20 years [225, 226].

Pathogenesis of PSC

The pathogenesis of PSC is multifactorial and is associated with genetics, immune development, environmental triggers, and physiological dysfunctions [227]. However, its precise mechanism is still unknown. HLA-B, IL-2, and G-protein coupled bile acid receptor 1(GPBAR1) were involved in disease pathogenesis [218]. The critical immune cells involved in inflammatory infiltration in PSC are T cells, which mediates the attack on bile duct epithelial cells, followed by macrophages and neutrophils [228]. Typical fibrotic cholangitis with irregular bile duct stenosis and scar formation might be mediated and triggered by HLA-restricted T cells, resulting in fibrogenic cytokines (such as transforming growth factor β) release [229]. Continuous stimulation of the inflammatory response leads to multiple interactions among infiltrating immune cells, hepatic stellate cells (leading to fibrosis and cirrhosis), and portal myofibroblasts (leading to multiple bile duct stenosis) [218, 229, 230]. The persistent inflammation and activation of vascular adhesion protein 1 (VAP1) in the liver lead to abnormal liver expression of mucosal addressin cell adhesion molecule-1(MAdCAM1) and CCL25 (normally expressed only in the colon) and recruitment of mucosal T cells to the liver [218].

A recent study found that approximately 70% of PSC patients develop inflammatory bowel diseases (IBDs), and 87% had ulcerative colitis suggesting the intestinal microbiome plays a role in initiating biliary immune response [229]. The close relationship between PSC and IBD indicates that they might share some common mechanisms in their pathogenesis [218].

Physiologically, the bile duct epithelium is generally protected by continuous hydrogen carbonate secretion and resistant to the inherent toxic bile. Immune-mediated injury affecting the integrity of bile duct epithelium, bile acid retention, and signal transduction are believed to exacerbate the disease [231, 232]. In addition, progressive periductal fibrosis in PSC might impair the oxygen and nutrient exchange between the peribiliary capillary plexus and the bile duct epithelium, resulting in ischemic injury [233]. This may also contribute to the structural damage of the bile duct in PSC.

Diagnosis of PSC

Based on their pathophysiology, PSC can be broadly divided into three types: a) symptoms caused by biliary obstruction and cholestatic liver failure; b) symptoms due to advanced chronic liver disease and cirrhosis of the liver (including gastrointestinal bleeding), and c) symptoms caused by inflammatory bowel diseases that complicate PSC. Symptoms caused by cholestasis include jaundice, cholangitis, pruritus, and abdominal pain. Malaise, jaundice, and pruritus are also presented in cases of advanced liver failure. These symptoms make differentiation by symptoms alone in early-stage challenging [227, 234].

The serum biochemical characteristics are usually cholestasis, mainly the increase of ALP level. Bilirubin and albumin levels are usually normal at an early stage, but with the progress of the disease, these levels may become increasingly abnormal. About 10% of PSC patients have elevated serum IgG4 levels, patients with elevated IgG4 levels tend to progress faster [235–237], and hypergammaglobulinemia is uncommon [238].

Autoantibodies can also be detected in patients with PSC, including antineutrophil cytoplasmic antibodies (ANCA), SMA, and ANA [239–242]. The most common serum antibody is perinuclear ANCA (p-ANCA), which can be found in about 93% of patients, followed by ANA (8–77%) and anti-SMA (0–83%) [243]. However, all these antibodies are not specific to PSC.

Imaging is a significant part of the diagnosis of PSC. Ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI) are usually used to eliminate biliary obstruction [227]. If no obstruction is found, the next step is usually cholangiography, including endoscopic retrograde cholangiopancreatography (ERCP) or MR cholangiopancreatography (MRCP) [244, 245]. MRCP is the preferred test because it is non-invasive, and it can show a typical "beaded" segment of the common bile duct due to the alternation of multifocal, short, circumferential stenosis with normal or slightly dilated segments of the common bile duct [3, 246] (Fig. 5).

Fig. 5.

Imaging of magnetic resonance cholangiopancreatography (MRCP) from primary sclerosing cholangitis (PSC). A Axial contrast-enhanced T1-weighted arterial phase MR imaging shows intrahepatic bile duct wall enhancement (arrow). B Coronal MRCP imaging showed circumferential stenosis of the right lobe bile duct (arrow). C Coronal MRCP imaging showed multiple localized stenosis and distal dilatation of the intrahepatic bile duct

If the imaging diagnosis is precise, liver biopsy is not necessary. Thus, liver biopsy is rarely used to diagnose PSC [247]. If AIH or small-duct PSC is suspected, A liver biopsy becomes necessary [247, 248]. Liver biopsy may also be helpful to evaluate the degree of interfacial hepatitis in PSC characterized by AIH or determine whether immunosuppressive therapy is needed [229], and evaluate the prognosis of PSC patients [240]. Cellular fibrosis and inflammatory cell infiltration around the bile duct are histopathologically characteristics of PSC. It is similar to onion and is called "onion skin fibrosis"[249], but the detection rate in biopsy specimens is shallow [229].

Treatment of PSC

For PSC patients with dominant strictures (common bile duct stenosis less than 1.5 mm or left and right hepatic duct stenosis less than 1 mm) [250], pruritus and/or cholangitis limited to extrahepatic and intrahepatic bile ducts, ERCP is recommended [251–255]. Balloon dilatation is recommended as the first-line endoscopic treatment [129, 256, 257]. Routine stenting is not required after dilation of dominant stenosis, while patients with severe stenosis may need short-term stenting [255, 257, 258].

There is no drug with a definite effect for PSC treatment, and the only definite treatment is liver transplantation for the end-stage of the disease [3]. End-stage liver disease [Model for End-stage Liver Disease (MELD) ≥ 15 points [226] or The United Kingdom Model for End-Stage Liver Disease (UKELD) over 49 scores [259]], recurrent cholangitis, decompensated liver disease, intractable pruritus, fatigue, and painful fractures that influence the quality of life are the most common indications for liver transplantation. In contrast, cholangiocarcinoma is usually a contraindication [260–263]. However, it was reported that about 20% of cases had recurrent PSC 5 years after liver transplantation [264].

Considering drug treatment, studies have shown that UDCA can improve the symptoms of cholestasis due to its protective effect on cytotoxic bile acids, bile acid-induced apoptosis, its antioxidant effect, and stimulating hepatobiliary secretion. However, evidence of the long-term benefits of UDCA for PSC therapy is unclear, and its use remains controversial. An appropriate dose of UDCA (17 – 23 mg/kg/day) could lead to moderate clinical, biochemical, and even histological improvements [227, 265]. On the other hand, the incidence of adverse events and the risk of colorectal cancer were higher in high-dose UDCA (28 – 30 mg/kg/day) [266, 267].

24-norursodeoxycholic acid (norUDCA) is a side chain shortened derivative of UDCA that bypasses the enterohepatic circulation, creating a 'bile liver shunt', resulting in reabsorption of bile acids by cholangiocytes and returning to the hepatocytes, secreting bicarbonate rich bile, and reducing bile toxicity to epithelial structures [268]. Animal experiments demonstrated that norUDCA had antiproliferative, antifibrotic, and anti-inflammatory effects [168, 269]. There was also a preliminary result from a phase III clinical experiment indicating that norUDCA might prevent liver fibrosis progression and reduce serum ALP levels (NCT03872921).

FXR, as a nuclear hormone receptor, can increase bile output and reduce bile acid reuptake in the liver and small intestine [270]. FXR agonists may be effective for PSC treatment. Studies have shown that OCA could reduce serum ALP levels [271] (NCT02177136), and the use of a nonsteroidal FXR agonist, Cilofexor, could produce a significant reduction in serum ALP levels with a low incidence of pruritus in patients with PSC [272].

PPAR agonists might also be a potential treatment. As mentioned above, Bezafibrate can induce and sustain normalization of serum ALP values in PBC with the added benefit of alleviating cholestatic pruritus [181, 182]. It has been shown that treatment with Bezafibrate could also reduce serum ALP value in patients with PSC [273] (NCT04309773).

ASBT can reabsorb 90% of bile acids transported to the small intestine. Thus, ASBT inhibitors could selectively block bile acid reuptake, decrease the total bile acid pool, and increase colonic transit [274] (NCT02061540), potentially alleviating cholestatic pruritus. As mentioned above, linerixibat is a small molecule inhibitor of ileal bile acid transporter with minimal absorption and selectivity. It can relieve itching in PBC patients. Although there is currently no definitive evidence that linerixibat can relieve pruritus, it is also one of our research directions in the future.

It was found that integrin αvβ6 level rising at the proliferating biliary epithelium could lead to tissue fibrosis through ductal reactions [275], and in animal experiments with PSC, a selective intervention targeting integrinαvβ6 could prevent liver fibrosis and tumorigenesis [276]. A current phase 2A clinical trial (NCT04480840) is ongoing to test the safety, tolerability, and pharmacokinetics of integrinαvβ6 inhibitor in patients with PSC. Eotaxin-2, also known as CeC motif chemokine ligand 24 (CCL24), and its receptor CCR3 have been shown to stimulate collagen synthesis and pulmonary fibrosis [277]. Therefore, anti-CCL24 treatment might also be an avenue to reduce inflammation and fibrosis. The clinical trial of humanized anti-CCL24 monoclonal antibody CM-101 (NCT04595825) is underway.

C–C chemokine receptor types 2 and 5 (CCR2 / CCR5) also play an essential role in inflammation and fibrosis. Studies have evaluated the efficacy and safety of the dual antagonist of CCR2 and CCR5 (Cenicriviroc) in the treatment of PSC (NCT02653625), which may be a potential novel therapeutic agent for PSC [278].

In addition, PSC might be associated with mucosal immune dysregulation and gut flora dysbiosis. Gut microbiome alterations in PSC can lead to alterations in bile acid metabolism, resulting in diminished intestinal barrier function [279]. Studies suggest that targeting the gut microbiome might change the course of the disease, thereby delaying or even halting disease progression [280]. Current studies targeting the gut microbiota in PSC included oral Vancomycin [281, 282] and fecal microbiota transplantation [283]. Other studies have shown that anti-TNFα antibodies (infliximab and adalimumab) might be considered to treat PSC [284, 285]. The drugs approved in clinical trials or pre-clinical PSC studies are listed in Table 4.

Table 4.

Registered trials for PSC cited in the text

Treatment	Mechanism	Current status	NCT number	Reference
UDCA	Cholagogic, promote bicarbonate secretion	Retrospective cohort study	-	227
24-norUDCA	Cholagogic, promote bicarbonate secretion	Phase III randomized, recruiting	NCT03872921	-
OCA	FXR agonists	Phase II randomized, completed	NCT02177136	271
Cilofexor	FXR agonist	Phase II randomized, completed	NCT02943460	272
Bezafibrate	PPAR agonist	Phase III randomized, recruiting	NCT04309773	273
LUM001	ASBT inhibitor	Phase II randomized, completed	NCT02061540	274
PLN-74809	Integrinαvβ6 inhibitor	Phase II randomized, recruiting	NCT04480840	-
CM-101	Anti-CCL24 monoclonal antibody	Phase II randomized, recruiting	NCT04595825	-
Cenicriviroc	CCR2 / CCR5 antagonist	Phase II randomized, completed	NCT02653625	278
Vancomycin	Change gut microenvironment	Phase I randomized, completed Phase III randomized, completed	NCT01322386 NCT01802073	281,282
Fecal microbiota transplantation	Change gut microenvironment	Pilot Clinical Trial	-	283
Infliximab	Immunosuppression	Retrospective cohort study	-	-
Adalimumab	Immunosuppression	Retrospective cohort study	-	-

ASBT apical sodium-dependent bile acid transporter, CCL CeC motif chemokine ligand, CCR C–C chemokine receptor, FXR farnesoid-X receptor, NCT national clinical trial, norUDCA 24-norursodeoxycholic acid, OCA obeticholic acid, PPAR peroxisome proliferator-activated receptors, UDCA ursodeoxycholic acid

For clinicians, the major challenges are preventing, treating PSC, and reversing the disease progress to avoid the development of end-stage liver disease, malignant tumor, and other diseases. The inconspicuous progress in the current treatment of PSC is much hindered by the insufficient and partial understanding of the disease mechanisms. With more studies directed to reveal how genetics, environmental, autoimmune, and physiological are orchestrated in the pathogenesis of PSC, the goal for early diagnosis, adequate follow-up, treatment, and malignant tumor screening, deciphering the specific pathogenic pathways, defining potential therapeutic targets, and effective therapies will be at hand.

Overlap syndromes

AIH–PBC overlap syndrome

AIH-PBC overlap occurs in 1–3% of patients with PBC and 7% of patients with AIH [286, 287]. PBC and AIH can occur simultaneously or sequentially in patients with PBC-AIH overlap syndrome. The 10-year progression rate of liver cirrhosis in AIH-PBC patients is 44 – 48% [288, 289], and the transplant-free survival rate is 52 – 92% [289–291].

Several is mainly treated with UDCA monotherapyd IAIHG scoring system, and the simplified version IAIHG scoring system [64, 70, 292, 293]. The Paris criteria are most commonly used to define the presence of PBC with features of AIH [292] and have been endorsed by EASL and AASLD (98) [294]. According to these criteria, at least 2 of 3 accepted items of PBC and AIH should exist, respectively. For AIH: (1) ALT > 5 × upper limit of normal (ULN); (2) IgG serum levels > 2 × ULN or presence of SMA; or (3) Moderate or severe periportal or periseptal lymphocytic piecemeal necrosis on histology. For PBC: (1) ALP > 2 × ULN or γ-GT > 5 × ULN; (2) Presence of AMA; (3) Florid bile duct lesion on histology. Histologic evidence of interface hepatitis is mandatory for the diagnosis.

Patients may develop overlap syndrome sequentially or may present simultaneously with both diseases. If the clinical course of AIH and PBC deviates from the classical course without known triggers, the overlapping syndrome should be suspected, such as hyperaminotransferases and hypergammaglobulinemia in patients with PBC and cholestasis in AIH patients. Sudden deterioration of liver function or poor response to treatment of previously well-controlled autoimmune liver disease should also raise the suspicion of overlap syndrome [295].

Studies have shown that UDCA may be effective in patients with an overlapping syndrome characterized by PBC. In the presence of severe interfacial hepatitis, UDCA needs to be combined with corticosteroids and/or other immunosuppressants (such as MMF and AZA) [124]. However, long-term studies on the prognosis of patients treated with UDCA and corticosteroids or combined immunosuppression are lacking.

AIH–PSC overlap syndrome

Overlapping syndrome AIH-PSC mainly occurs in young people and children. The diagnostic criteria of AIH-PSC overlapping syndrome are not clear. AIH-PSC overlap syndrome happens in less than 6% of PSC patients [296–299]. In adult AIH patients, the prevalence of AIH-PSC depends on the presence (44%) or absence (8%) of comorbid IBD [5]. This syndrome is more common in children and is called autoimmune sclerosing cholangitis (ASC) with up to 50% of AIH children having cholangiographic features of PSC [7]. At present, UDCA combined with corticosteroids (with or without AZA) is the most common treatment [300, 301].

Other Overlap Syndrome of ALDs

Overlapping PBC-PSC [302–308] and AIH-PBC-PSC [305, 309] have also been reported in recent years, but there is no standardized diagnostic standard for these diseases. PBC-PSC usually has the histological features of cholestasis, AMA positive and/or histological characteristics of PBC, and the cholangiography and/or histological features of PSC. In addition to the above characteristics, AIH-PBC-PSC also has biochemical, immunological, and/or histological evidence of AIH. PBC-PSC is mainly treated with UDCA monotherapy [303, 304]. AIH-PBC-PSC was treated with prednisolone and AZA [305], UDCA combined with prednisolone /budesonide, and AZA [307].

Conclusions

Among liver diseases, ALDs predominantly affecting women and can develop at any age is a group of chronic liver diseases with unknown etiology. They should be seriously considered for every patient with unexplained acute or chronic hepatitis and/or liver cirrhosis. The mechanisms responsible for the sex differences in autoimmune diseases are largely unknown. In addition to genetic predisposition, recent studies have suggested that levels of sex hormones, epigenetic alterations on the X chromosome, increased exposure to household chemicals and environmental pollutants in westernized countries may be involved in gender bias in ALDs [310].

The clinical manifestations and liver biochemistry of ALDs are homogeneous.In the vast majority of patients, although the histological results are not necessarily specific for diagnosing ALDs, liver biopsy is helpful for the diagnosis and evaluation of disease severity. Thus, more defined and unified diagnostic criteria for diagnosing ALDs are required. In terms of treatment and the clear effect of UCDA, sometimes with OCA on most PBC, of glucocorticoid and few immunosuppressive agents on the majority of AIH, there is no effective discovery in the treatment of PSC and the refractory cases of the other two diseases. Recently, increasing attention has been paid to antigen-specific immunotherapy for autoimmune diseases [311]. For ALDs patients (especially AIH and PBC patients with autoimmune antibodies), antigen-specific immunotherapy might have potential therapeutic value in the future [312].

Increasing efforts are directed towards understanding the mechanisms of diseases in ALDs. Data from genetics and epigenetics studies are expected to refine the scope on the prioritization of targeted molecules and pathways for identifying new biomarkers and immunological checkpoints for the diagnosis, prevention, and treatment of ALDs to tailor treatment options to improve prognosis further.

For now, there are many challenges in the development of new therapies for ALDs, including the complexity of trial design, patient recruitment, ethical approval, and funding, which are also increased by the prevalence of COVID-19. More profound research on new drugs, such as immunomodulators, anti-inflammatory, antifibrotic drugs, or drugs targeting the gut microbiota will provide more options for treating ALDs. The ideal treatment for ALDs is a combination of different drugs that target different pathogenic mechanisms. Although data on reliable endpoints are not available in most cases, we hope that ongoing/future studies in defining the efficacy and safety of promising targeted therapies will further direct the path towards individualized medicine of high efficacy and minimal side effects for a better quality of life. Our goal is to cure ALDs, away from lifelong corticosteroids and/or immunosuppressant therapy. Although seemingly distant, this can be achieved through a concerted, collaborative effort between patients, clinicians, wet bench scientists, and the pharmaceutical industry.

Abbreviations

APCs

Antigen-presenting cells

CXCR3

C-X-C motif chemokine receptor-3

CTL

Cytotoxic T cell

DCs

Dendritic cells

HBV

hepatitis B virus

HCV

hepatitis C virus

IFN-γ

Interferon-γ

IL

Interleukin

LSEC

Liver sinusoidal endothelial cells

MHC

Major histocompatibility complex

NK

Natural killer cell

TCR

T cell receptor

TGF-β

Transforming growth factor β

Th

T helper

TNF

Tumor necrosis factor

Treg

Regulatory T cell

Author Contribution

Yangfan Chen, Ruofei Chen, and Haiyan Li wrote the main manuscript text and Yangfan Chen prepared figures 1-5. All authors reviewed the manuscript.

Funding

The grant supporting this program was the National Natural Science Foundation of China [NO. 81871296].

Data availability

No datasets were generated or analysed during the current study.

Declarations

This study did not involve human or animal subjects, and thus, no ethical approval was required.

Competing interests

The authors declare no competing interests.