Rapamycin nanoparticles suppress autoreactive lymphocytes and reduce anti-mitochondrial antibodies in primary biliary cholangitis: Mechanisms and implications

Abstract

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by the selective destruction of intrahepatic small bile ducts, primarily by infiltrating lymphocytes, and has limited therapeutic options. A growing body of evidence suggests that nanoparticles encapsulating rapamycin (ImmTOR) can suppress autoreactive lymphocytes and reduce inflammatory cytokine levels in various autoimmune diseases. In a recent study, Yang et al investigated the therapeutic effects of ImmTOR in a mouse model of PBC. ImmTOR treatment reduced the expression and number of CD4+ T cells, CD8+ T cells, and B cells isolated from the liver and spleen, improved liver inflammation and enzyme levels, and was associated with a concomitant decrease in anti-mitochondrial antibody levels. In this editorial, we highlight the significance of these findings, focusing on the potential mechanisms by which ImmTOR suppresses hepatic autoreactive T cells and reduces anti-mitochondrial antibody levels, ultimately improving liver pathology, through pathways such as mammalian target of rapamycin inhibition and autophagy restoration. We also offer a perspective on future research directions for PBC in both animal models and in vitro studies.

Key Words: Primary biliary cholangitis; Rapamycin nanoparticles; Nanoparticles encapsulating rapamycin; Apoptosis; Autophagy; Lymphocytes; Autoimmune disease

Core Tip: Primary biliary cholangitis (PBC) is an autoimmune disease with limited therapeutic options. A recent study using a mouse model of PBC found that nanoparticles encapsulating rapamycin treatment reduced the levels of autoreactive T cells and B cells in the liver and spleen, improved liver inflammation and enzyme levels, and was associated with a concomitant decrease in anti-mitochondrial antibody levels. These findings indicate that nanoparticles encapsulating rapamycin may improve liver pathology in the PBC mouse model through mammalian target of rapamycin inhibition and potential autophagy restoration, suggesting it could represent a novel therapeutic option for this chronic disease.

INTRODUCTION

Primary biliary cholangitis (PBC) is a cholestatic disease that is prevalent in Japan and China, with limited therapeutic options. The molecular mechanisms underlying its pathology are not yet fully understood[1-4]. PBC is initiated by viral or xenobiotic-induced apoptosis of bile duct cells (BDCs) within the intrahepatic biliary ductules. It is aggravated by the infiltration of autoreactive lymphocytes into the affected ductules and the presence of elevated anti-mitochondrial antibodies (AMA), ultimately resulting in biliary cirrhosis[1]. The apoptotic destruction of BDCs is enhanced by infiltrating CD4+ and CD8+ T cells, leading to secondary damage of hepatocytes and the production of inflammatory cytokines. Meanwhile, mitochondrial antigens pyruvate dehydrogenase complex E2 subunit within apoptotic BDCs stimulate the production of AMAs by activated autoreactive B lymphocytes and plasma cells. Therefore, PBC is considered an autoimmune disease[2].

AUTOPHAGY MECHANISMS AND ROLES IN PBC

Recent studies suggest that dysfunctional autophagy, followed by cellular senescence in BDCs, leads to the abnormal expression of mitochondrial antigens and contributes to the autoimmune pathogenesis of PBC[3,4]. Macroautophagy (commonly referred to as autophagy) is an essential cellular process responsible for the clearance of abnormal proteins, damaged mitochondria, and other cytoplasmic components, particularly in response to stressors such as inflammation, oxidative stress, and xenobiotics[5]. Autophagy is activated through inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) and subsequent activation of AMP-activated protein kinase signaling, resulting in the formation of beclin-1-mediated autophagosomal membranes. These membranes mature into autophagosomes that sequester cellular cargo for clearance by lysosomes[6]. In addition, autophagy is required for the clearance of apoptotic cells, preventing autoimmune diseases such as systemic lupus erythematosus[7].

IMMUNOSUPPRESSIVE AND AUTOPHAGY-MODULATING EFFECTS OF RAPAMYCIN NANOPARTICLES AS A THERAPY FOR AUTOIMMUNE DISEASES

Nanoparticles encapsulating rapamycin (ImmTOR) administered intravenously increased the therapeutic window of engineered interleukin-2, promoted the expansion of antigen-specific regulatory T (Treg) cells, and reduced inflammatory T cells in animal models, indicating its utility as a treatment for various autoimmune diseases, including type I diabetes, graft vs host disease, and PBC[8]. In previous studies, rapamycin reportedly suppressed autoreactive T and B cells[9,10] and reduced autoimmune dacryoadenitis in Sjögren’s syndrome mouse models via activation of autophagy and suppression of T cells[11]. Moreover, a recent in vitro study found that rapamycin upregulated autophagy in stem cells, secreting transforming growth factor-beta 1 and promoting Tregs to suppress inflammatory CD4+ T cells[12].

RAPAMYCIN NANOPARTICLES SUPPRESS AUTOREACTIVE LYMPHOCYTES AND REDUCE AMA IN A MOUSE MODEL OF PBC

Using flow cytometry analysis, Yang et al[13] found that intraperitoneal injection of rapamycin nanoparticles into a mouse model of PBC led to a significant reduction in autoreactive CD4+ and CD8+ T cells, as well as B cells isolated from the liver. Improved liver inflammation and enzyme levels were also recorded in the mice, along with a concomitant decrease in anti-AMA levels using biochemical analyses and enzyme linked immunosorbent assay (Figure 1). These results strongly support the suppressive effect of ImmTOR on B cell activity and autoantibody production against mitochondria, and indicate a durable immunosuppressive effect throughout disease progression[14]. A significant reduction in CD8+ T cells (P < 0.001) and B cells (P < 0.05) was observed in the liver, but no change in CD4+ T cells isolated from the spleen[13]. This organ-specific modulation aligns with selective hepatic immune tolerance, supported by existing literature on nanoparticle uptake by liver-resident immune cells[15]. These findings were statistically rigorous and biologically consistent with the hypothesized mechanism of ImmTOR promoting antigen-specific immune tolerance in the liver microenvironment[16,17]. Overall, this study provides compelling scientific evidence that ImmTOR significantly reduced autoimmune-mediated hepatic inflammation in a murine model of PBC, warranting its potential as a PBC therapy and basis for further translational research.

Figure 1 Mechanisms underlying the hepatoprotective effects of rapamycin nanoparticles in primary biliary cholangitis[13]. A: Intraperitoneal injection of nanoparticles encapsulating rapamycin (ImmTOR) in primary biliary cholangitis (PBC) mice resulted in inhibition of mechanistic target of rapamycin signaling, suppression of autoreactive B and T lymphocytes, reduction of anti-mitochondrial antibodies, and decreased levels of pro-inflammatory cytokines. These effects ultimately led to improvements in liver pathology and function. Notably, ImmTOR may also induce upregulation of autophagy in hepatic cells and bile duct cells (BDCs), thereby enhancing autophagy-mediated clearance of apoptotic BDCs and contributing to reduced inflammation and anti-mitochondrial antibodies production. Additionally, ImmTOR may promote the expansion of regulatory T cells, which exert anti-inflammatory effects; B: Inflammatory cell infiltration around damaged BDCs in the liver of untreated PBC mice (indicated by arrows); C: Liver of an ImmTOR-treated PBC mouse with reduced inflammation, the green dotted arrows indicate mechanisms proposed for future research. B and C: Citation: Yang YS, Li XR, Wang ZM, Zheng L, Li JL, Cui XL, Song YB, Ma JJ, Guo HF, Gao LX, Zhou XH. Effect of rapamycin nanoparticles in an animal model of primary biliary cholangitis. World J Hepatol 2025; 17: 104073. Copyright© The Authors 2025. Published by Baishideng Publishing Group Inc. This article is an open-access article. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/. IP: Intraperitoneal; ImmTOR: Nanoparticles encapsulating rapamycin; PBC: Primary biliary cholangitis; mTOR: Mechanistic target of rapamycin; Treg: Regulatory T; AMA: Anti-mitochondrial antibodies; BDC: Bile duct cell.

To further strengthen the study, immunohistochemistry techniques could be utilized to characterize the localization and expression of CD4+, CD8+, T cells, B cells, and Tregs within the spleen and liver tissues, as well as to elucidate their associations with hepatocytes and BDCs[7-9]. Additionally, using the nick-end labeling method, specific immunosuppressive mechanisms mediated by ImmTOR, its potential role in enhancing lymphocyte apoptosis, and apoptotic BDCs can be investigated. Such analyses would contribute valuable mechanistic insights into the immunopathogenesis of PBC[1,2,7]. In the discussion section, Yang et al[13] failed to clearly explain the mechanisms behind ImmTOR-activated autophagy for PBC therapy, nor did they substantiate this claim with appropriate references. Several mechanisms support the notion that restoration of autophagy may constitute a therapeutic strategy in PBC. First, autophagy activation in autoreactive lymphocytes, hepatic macrophages, and hepatocytes has been shown to suppress the production of pro-inflammatory cytokines[5,7,17,18]. Second, autophagy activation in BDCs may prevent apoptosis, thereby reducing the initiation of PBC[5,7]. Importantly, autophagy may mediate the clearance of mitochondrial antigens and apoptotic BDCs, thereby limiting the generation of AMAs and suppressing autoimmune responses[1,7,19,20]. Further investigation into the role of autophagy in ImmTOR-induced hepatoprotection in PBC is warranted, particularly through the use of established autophagy markers such as beclin-1 and LC3-II[5-7]. Previous studies in animal models of autoimmune diseases have shown upregulation of these markers in affected organs[21,22], potentially supporting the immunosuppressive effects observed with rapamycin-loaded nanoparticles in the study by Yang et al[13].

While Yang et al[13] proposed that ImmTOR modulates immune cell populations through inhibition of the mechanistic target of rapamycin (mTOR) pathway, their study did not include a direct assessment of the phosphorylation status of key downstream effectors, such as S6 kinase or eukaryotic translation initiation factor 4E-binding protein 1. A potential mechanism involves the inhibition of the mTORC1, as seen with rapamycin and related mTOR inhibitors, leading to S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 dephosphorylation. As a result, mTORC1-mediated suppression of autophagy-related proteins is alleviated, thereby promoting autophagic activity[23]. However, the mechanisms underlying autophagy dysregulation in relation to gene expression and key cellular pathways involved in PBC development remain insufficiently characterized. Furthermore, there is a lack of direct molecular evidence supporting the proposed mechanism of mTOR suppression in immune cells, causing the investigation to lack substance. Importantly, Yang et al[13] did not address the key parameters associated with the administration of ImmTOR, including dosage, duration, and route of administration (i.e., intraperitoneal vs intravenous). Additional limitations of the study include the small sample size, grouping and potential gender bias present in the study. A total of 16 mice (8 per group) were used in this study, which represents a relatively limited sample size for conducting robust multivariate analysis across flow cytometry, enzyme linked immunosorbent assay, and histopathological evaluations[24]. The cohort of 12 females and 4 males presents an unjustified ratio and does not follow established guidelines for gender distribution design in preclinical studies[25]. Dose-response and pharmacokinetic data were also absent from the study, with only one unsubstantiated dose of ImmTOR tested. Dose-ranging and toxicity assessments were markedly lacking, and the design of the control group was insufficient. The inclusion of free and nanoparticle-loaded rapamycin at varying doses would provide a more comprehensive assessment of liver uptake and formulation-specific effects[26]. The 12 weeks study lacked any post-treatment follow-up assessment for relapse or long-term effects. Given that PBC often relapses after withdrawal of immunosuppressive therapy, durability of the treatment is essential for clinical translation[27]. The lack of detailed histopathological investigation in the study, including types of inflammatory cells, presence of hyperplasia, and other structural abnormalities, such as bile duct ectasia, is evident. It is unclear whether histological scoring was performed using a validated system or if assessments were blinded, raising concerns about potential observer bias[8,9,11,28]. While Yang et al[13] provide promising preliminary evidence that ImmTOR may attenuate immune-mediated liver injury in PBC, the study lacks mechanistic validation, proper controls, nanoparticle characterization, safety data, and detailed histopathologic detail, limiting its translational impact. Additionally, redundancy in the methods section (page 6) and language errors detract from the manuscript’s clarity and quality, underscoring the need for professional editing.

PERSPECTIVES FOR FUTURE RESEARCH ON PBC

A key question that remains unresolved in the study is the ability of ImmTOR to restore autophagy in PBC. In vitro and animal studies are necessary to elucidate the molecular mechanisms underlying PBC, specifically the role of autophagy-related gene dysfunction in this disease. The use of various in vitro and animal models would provide more insight into the cellular targets, hepatic uptake, and therapeutic efficacy of the rapamycin nanoparticles. Additionally, key parameters such as dosing, particle size, route of administration, treatment duration, and post-treatment follow-up for ImmTOR therapy require rigorous validation to support clinical translation. Future studies should incorporate blinded histological scoring, include both sexes, and utilize relevant control groups to enhance the reliability of the results. Figure 1 summarizes the hepatoprotective, immunosuppressive, and autophagy-modulating pathways in PBC upon exposure to rapamycin nanoparticles, as reported by Yang et al[13], as well as mechanisms proposed for future investigations.

CONCLUSION

PBC is a chronic autoimmune disease characterized by the destruction of BDCs within intrahepatic bile ductules, accompanied by the infiltration of autoreactive lymphocytes and the production of AMA. ImmTOR has been shown to reduce hepatic infiltration of autoreactive lymphocytes, decrease pro-inflammatory cytokine levels, and lower AMA levels in a mouse model of PBC. These effects were associated with improvements in liver inflammation, hepatic enzyme levels, and histopathological features. The hepatoprotective properties of ImmTOR are attributed to both the immunosuppressive effects of rapamycin and potential autophagy activation. However, further studies using in vitro systems and animal models of PBC are necessary to optimize the strategies for ImmTOR administration, including nanoparticle size, route of delivery, and dosage, as well as identification of the specific hepatic cell populations responsible for nanoparticle uptake. Further experimental work on activation of the autophagy gene and enhanced autophagic flux by ImmTOR in the treatment of PBC is also warranted.