Immunomodulatory effects of tacrolimus-loaded lipid-core nanocapsules in autoimmune hepatitis

Abstract

Liver damage in autoimmune hepatitis (AIH) is perpetrated by T-effector lymphocytes that are not adequately restrained by regulatory T cells (Tregs). The goal of AIH treatment is to control inflammation and induce disease remission through administration of immunosuppressive drugs including corticosteroids, azathioprine, and, in difficult-to-treat cases, mycophenolate mofetil or tacrolimus (TAC). Despite TAC being a potent immunosuppressant, its use has been hampered by a narrow therapeutic window and systemic toxicity.

In this study, we have tested the effects of lipid-core nanoformulations encapsulating TAC (NC-TAC) as a novel drug delivery system that would enable potentially favorable immunomodulatory effects compared with unencapsulated TAC. NC-TAC properties were assessed in vitro in CD4 T cells and Tregs isolated from the peripheral blood of AIH patients and controls, and in vivo through a model of T cell-mediated liver injury induced by Concanavalin-A (Con-A) in NOD/scid/gamma mice, pre-emptively reconstituted with human CD4 T lymphocytes.

Compared to unencapsulated TAC, NC-TAC favored a regulatory phenotype in CD4 T cells of AIH patients, enhanced the suppressive function and preserved AIH Treg phenotype in the presence of an inflammatory stimulus. Systemic administration of NC-TAC ameliorated liver injury in vivo, as indicated by decreased ALT levels, reduced lymphocyte infiltration on histology, and an increased frequency of intrahepatic CD4⁺FOXP3⁺ lymphocytes.

NC-TAC could therefore be considered as a novel drug delivery system to be possibly explored for the treatment of AIH, having a beneficial immunomodulatory profile and effectively favoring Treg immune responses.

Graphical Abstract

1. Introduction

Autoimmune hepatitis (AIH) is a chronic and progressive hepatopathy that mainly affects females and is characterized by hypergammaglobulinemia, positivity for serum autoantibodies and evidence of interface hepatitis on histology [1, 2]. AIH typically follows a relapsing-remitting course, which, despite immunosuppressive treatment, may result in end-stage liver disease requiring transplantation [3, 4]. Liver damage in AIH is mainly driven by T-helper type 1 (Th1) and T-helper type 17 (Th17) cells [5-7] that are not adequately controlled by regulatory T cells (Tregs) [8-10]. In AIH, dysfunctional Tregs play an important permissive role by facilitating effector cell immunity and perpetuating liver injury.

Immunosuppression remains at large the mainstay of AIH treatment and relies on the administration of broad-spectrum immunosuppressive agents, such as corticosteroids and azathioprine. Alternative regimens consist of mycophenolate mofetil [11, 12] and calcineurin inhibitors like cyclosporine and tacrolimus (TAC) [13-15]. These drugs induce biochemical remission in a large proportion of patients [16]. In their current formulation, however, they are often associated with significant side effects, impacting patients’ quality of life and adherence to treatment. TAC is a potent immunosuppressant used in various autoimmune conditions because of its ability to control T-cell activation [17]. Its clinical use, however, has been hampered by a narrow therapeutic window and significant systemic toxicity [18]. Recent advancements in nanotechnology offer promising solutions to these challenges by enhancing drug delivery and targeting capabilities [19-23].

Lipid-core nanocapsules (NC) have emerged as an innovative drug delivery system that can improve the pharmacokinetic and pharmacodynamic profiles of encapsulated agents. These NC can protect drugs from degradation, enhance their bioavailability, and provide controlled release, thereby increasing therapeutic efficacy while reducing adverse effects [24]. In this context, TAC encapsulation within lipid-core nanocapsules (NC-TAC) represents a novel approach to enhance the therapeutic potential of this immunosuppressant for autoimmune diseases, including AIH. In this study, we tested the efficacy of NC-TAC in vitro in peripheral blood-derived CD4 T cells and Tregs of AIH patients and controls, and in vivo using an experimental model of liver injury in humanized mice.

We report that in AIH, NC-TAC boosts regulatory immune responses, as reflected by increased Treg suppression and preservation of Treg immunophenotype in vitro, and ameliorates liver damage in vivo. These data indicate that NC-TAC might represent a preferable option in the setting of AIH when compared to unencapsulated TAC because of its favorable immunomodulatory profile.

2. Materials and Methods

2.1. Materials

TAC was obtained from Fagron (São Paulo, Brazil), while Poly(ε-caprolactone) (PCL) with a molecular weight of 80 kg/mol was purchased from Perstorp UK Limited (Warrington, United Kingdom). Caprylic and capric triglyceride (CCT) were purchased from Delaware (Porto Alegre, Brazil) while polysorbate 80 was sourced from Henrifarma (São Paulo, Brazil). Sorbitan monostearate (Span 60^®) was from Sigma Aldrich (São Paulo, Brazil), and from Labsul (Porto Alegre, Brazil). All other chemicals and solvents that met analytical or pharmaceutical standards were utilized without further treatment.

2.2. Development of lipid-core nanocapsules

Nanosuspensions were obtained through self-assembly using solvent displacement technique. In brief, the organic phase, consisting of PCL (0.1 g), Span 60^® (0.04 g), a mixture of caprylic/capric triglyceride (160 μL), and TAC (0.008 g), was dissolved in acetone at 40°C and slowly injected with moderate magnetic stirring into an aqueous solution containing polysorbate 80 (0.08 g). The resulting translucent solution was stirred for 10 minutes. Subsequently, acetone was evaporated, and the suspension concentrated under reduced pressure to a final volume of 10 ml. These nanosuspensions were denoted as NC (control, without TAC) and NC-TAC (loaded with TAC, 0.80 mg/mL). All formulations were prepared in triplicate batches. No organic solvent was present in the final formulation at the time of in vitro or in vivo administration. Unencapsulated TAC was dissolved in water containing polysorbate 80 at the same mass fraction as in the nanocapsule dispersions. TAC vehicle (i.e., water plus polysorbate 80 at the same mass fraction) was used for in vitro and in vivo experiments.

2.3. Characterization of lipid-core nanocapsules

NC and NC-TAC underwent evaluation for size, polydispersity index, zeta potential, pH, drug content (NC-TAC), and encapsulation efficiency (NC-TAC). Laser diffraction analysis was conducted using a Mastersizer 2000 (Nano ZS; Malvern Instruments, UK) with direct insertion into a Hydro2000 unit filled with distilled water under constant agitation. Dynamic light scattering (Zetasizer Nano ZS; Malvern Instruments) was used for size distribution and polydispersity index determination after diluting samples in ultrapure water. Zeta potential was measured using a Zetasizer Nano-ZS ZEN 3600 model (Malvern Instruments Ltd., UK) after dilution in 10 mM NaCl solution. pH was determined directly with a potentiometric probe (B474 Micronal, Brazil).

For drug content assessment, HPLC-UV analysis was performed using a C18 Reversed-phase column (4.6×250 mm, 5 μm) with an acetonitrile:water:phosphoric acid mobile phase. Ultrafiltration centrifugation was utilized to quantify unencapsulated TAC. Encapsulation efficiency was calculated as the difference between total and unencapsulated TAC concentrations. All measurements were performed in triplicate batches.

$$EE\%=\frac{(Ct-Cf)}{Ct}x100$$ Eq. (1)

Here, C_t is the drug content and C_f corresponds to TAC_free in the ultrafiltrate.

2.4. Subjects

Peripheral blood mononuclear cells (PBMCs) were isolated from twenty-four patients with anti-nuclear antibody (ANA) and/or smooth muscle antibody (SMA) positive AIH. At the time of disease presentation, patients had histopathological features of interface hepatitis and were all positive for ANA and/or SMA autoantibodies. Seventeen patients were studied during remission (i.e., normal transaminase levels); whereas seven were studied during an episode of relapse. Immunosuppressive regimens included azathioprine (25-75 mg/day); mycophenolate mofetil (500 mg-3 g/day), alone or in combination with prednisone (4.5-9 mg/day); and budesonide (3 mg/day), alone or in combination with mycophenolate mofetil. None of these patients were treated with TAC. Two patients were not on immunosuppression at the time of study. Of these two, one was in remission and underwent treatment withdrawal because of normal transaminase levels for more than two years; the other patient was studied during an episode of relapse due to low drug compliance. Demographic, clinical and laboratory data of all patients enrolled in this study are presented in Supplemental Table 1. Eleven age and sex matched healthy individuals served as controls. The study received IRB approval at Beth Israel Deaconess Medical Center, Boston, MA (protocol # 2018P000019). Written consent was obtained from all study participants prior to inclusion in the study.

2.5. Isolation of PBMCs

PBMCs were isolated from 20 mL peripheral blood collected in EDTA tubes. Cells were separated using Ficoll-Paque (GE Healthcare, Chicago, IL). After isolation, PBMCs were washed twice with 1× phosphate buffered saline (PBS). Cell viability was verified using Trypan Blue and exceeded 98% in all cases.

2.6. Cell purification

Total CD4 T cells were purified from PBMCs by negative selection according to the manufacturer’s recommendations (Miltenyi Biotec, San Diego, CA). The purity of CD4 T cells as tested by flow cytometry was higher than 92%, as in our previous work [25] (Supplemental Fig. 2). Tregs were isolated by immunomagnetic beads as CD4⁺CD25^highCD127⁻ cells according to the manufacturer’s instructions (Miltenyi Biotec). Treg purity consistently exceeded 92%, as we have previously reported [26] (Supplemental Fig. 3). Purified CD4 T cells and Tregs were subsequently exposed to NC, NC-TAC, TAC or vehicle at a concentration equivalent to 20 nM TAC for 24 hours. When testing immunophenotype in the presence of a pro-inflammatory challenge, Tregs were obtained following polarization of naïve CD4 T lymphocytes in the presence of IL-2 (100 ng/ml), TGF-β (10 ng/ml) and Dynabeads Human T activator CD3/CD28 for T cell expansion (bead/cell ratio: 1/2) for five days, due to the higher cell yield required for this experiment.

2.7. Flow cytometry

The frequency of FOXP3⁺, RORC⁺, IL-10 ⁺, IL-17A⁺ and IFNγ⁺ lymphocytes within the CD4 T cell subset was measured by flow cytometry. After a 24-hour exposure to NC, NC-TAC, TAC or vehicle, cells were harvested, washed with 1× PBS and stained using anti-human CD4 (clone # OKT4, Biolegend, San Diego, CA) antibodies. Following fixation and permeabilization using the eBioscience FOXP3/Transcription Factor Staining Set (Thermo Fisher Scientific, Waltham, MA), cells were stained with anti-human FOXP3 (clone # PCH101, Thermo Fisher Scientific), IL-10 (clone # JES3-9D7, Biolegend), IL-17A (clone # BL168, Biolegend), IFNγ (clone # B27, BD Pharmingen, Franklin Lakes, NJ) and RORC (clone # AFKJS-9, Thermo Fisher Scientific) antibodies. To assess the percentage of apoptotic cells after treatment with NC, NC-TAC, or TAC, CD4 T cells were washed twice with 1× PBS. The cell pellet was then resuspended in a mixture consisting of 1× binding buffer (Biolegend), propidium iodide (PI, Biolegend), and Annexin V (Biolegend). A total of 2×10⁵ cells per sample were utilized. Cells were then incubated for 15 minutes in the dark at room temperature. Annexin V positivity marked early-stage apoptotic cells, while positivity for both Annexin V and PI indicated late apoptotic cells. Cells were acquired on a CytoFLEX LX Flow Cytometer (Beckman Coulter, Pasadena, CA) and analyzed using FlowJo 2 software (version 10, TreeStar, Ashland, OR). Positively stained cell populations were gated based on unstained and single stained controls. Compensation was adjusted based on the fluorescence-minus-one method.

2.8. qRT-PCR

Expression of FOXP3, RORC, IL-10, IL-17A and IFNγ was determined by qPCR, as previously described [27], following extraction of total RNA using TRIzol reagent (Thermo Fisher Scientific). mRNA was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer’s instructions. Samples were run on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA), and the results were analyzed by matched software and expressed as relative quantification. Relative gene expression was determined after normalization to human ACTB or GAPDH. Primer sequences are included in Supplemental Table 2. Prime Time qPCR primers for the detection of human FOXP3, ACTB and GAPDH were pre-designed by and purchased from Integrated DNA Technologies (Coralville, IA).

2.9. Proliferation assay

Total CD4 T cells or Tregs were seeded into 96-well plates at 2 × 10⁵ cells/well and treated with NC, NC-TAC, TAC or vehicle. Cell proliferation was assessed following exposure to Dynabeads T activator CD3/CD28 [bead/cell: 1/2] and IL-2 at 100 ng/mL for 3 days. Proliferation was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Twenty μl Cell Titer Aqueous One Solution Reagent was added to each well. After 60 minutes at 37°C and 5% CO₂, absorbance was recorded at 490 nm. Absorbance values for each condition represent the average of triplicates.

2.10. Suppression assay

The ability of Tregs to suppress was assessed in co-culture experiments in which Tregs exposed to NC, NC-TAC, TAC or vehicle were added at a ratio of 1/8 to autologous CD4⁺CD25⁻ target cells [10], which were also isolated using immunomagnetic beads (Miltenyi Biotec). Parallel cultures of CD4⁺CD25⁻ cells without Tregs were performed under identical conditions. Responder cells were activated using IL-2 (30 IU/ml) and Dynabeads Human T activator CD3/CD28 (bead/cell ratio: 1/2) for three days. Proliferation of CD4⁺CD25⁻ cells, in the absence or presence of Tregs, was determined using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega), as indicated above. Absorbance values for each condition represent the average of triplicates.

In an additional set of experiments, Tregs were exposed to a pro-inflammatory challenge consisting of IL-6 (0.04 μg/ml) and IL-1β (0.01 μg/ml) [25] for thirty-six hours, in the absence or presence of NC, NC-TAC or TAC added for the last twenty-four hours of culture. Expression of FOXP3, RORC, IL-10 and IL-17A was measured afterwards.

2.11. Induction and assessment of liver injury

Acute liver injury was induced by administration of Concanavalin-A (Con-A) in NOD/scid/gamma mice that had been pre-emptively transferred with human CD4 T cells, as we have previously reported [25, 26]. Six-week-old female NOD/scid/gamma mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and kept under pathogen-free conditions. Mice were reconstituted using 2-3 × 10⁶ CD4 T cells, obtained from the peripheral blood of one healthy blood donor. Three weeks later, mice were bled and checked for human chimerism [25, 26]. The rate of human chimerism across different treatment groups was comparable. Mice achieving more than 10% human chimerism were randomly assigned to treatment groups. Treatment was administered in a blinded fashion. NC, NC-TAC, TAC or vehicle were administered intraperitoneally once a day for three days. NC-TAC and TAC groups received doses equivalent to 6 mg/kg/day of TAC [19]. On day 4, mice were given Con-A (Millipore Sigma) at 20 mg/kg i.v. and sacrificed four hours later. Mice reconstituted with human CD4 cells but unexposed to Con-A served as controls. At the harvest, blood was withdrawn from the vena cava and spleen and liver were collected from each recipient. Serum ALT was measured using ALT (SGPT) Kinetic Method (Teco Diagnostics, CA, US) (assay normal values: 3 - 35 U/L). Lymphocytes were isolated from spleen using Buffer EL (Erythrocyte lysis buffer) (Qiagen, Germantown, MD). Intrahepatic lymphocytes were obtained as previously described [25, 28]. The phenotype of splenic and liver infiltrating lymphocytes was assessed by flow cytometry. Staining was carried out using anti-human CD3 (clone # OKT3, Biolegend), CD4 (clone # OKT4, Biolegend), IL-10 (clone # JES3-9D7, Biolegend), IL-17A (clone # BL168, Biolegend), and IFNγ (clone #B27, BD Pharmingen) antibodies. Expression of FOXP3 was determined using the eBioscience FOXP3/Transcription Factor staining buffer set (Thermo Fisher Scientific) followed by staining with anti-human FOXP3 (clone # PCH101, Thermo Fisher Scientific). Flow cytometry analysis was carried out, as indicated above. The animal protocol was approved by the Animal Care and Use Committee at BIDMC, Boston (protocol # 052-2024). Animal experiments were independently repeated three times.

2.12. Immunohistochemistry staining

Paraffin-embedded liver tissue sections were subjected to antigen retrieval [29]. Six μm tissue sections were then stained with hematoxylin and eosin and examined by a pathologist for evidence of liver injury. Modified Ishak Histological Activity Index (mHAI) score was calculated along with the lymphoplasmacytic infiltration score that was determined as previously reported [30].

Sections were also stained with anti-human CD3 antibody to verify reconstitution with human CD3 lymphocytes at the tissue level. To this end, sections were incubated with 1/1,000 goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Following treatment with Vectastain Elite ABC kit (Vector Laboratories), ImmPACT DAB (Vector Laboratories) was applied, and sections were examined by light microscopy.

2.13. Statistical analyses

Normality of variable distribution was assessed by Kolmogorov-Smirnov Goodness of fit test. Comparisons were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. P≤0.05 was considered significant. Statistical analysis was performed using GraphPad Prism, version 9.20 (GraphPad Software, San Diego, CA). Data are represented as either violin plots (with median and quartiles shown) or box and whisker plots.

3. Results

3.1. Development and characterization of lipid-core nanocapsules

Analysis of particle size by laser diffraction confirmed a monomodal distribution with exclusive nanoparticles. The diameter distribution displayed a uniform pattern, with a volume-weighted mean diameter (D[4.3]) of 185 ± 10 nm. The inclusion of the drug did not have a discernible impact on the diameter of the lipid-core nanocapsules, and polydispersity (span) values were below 1.6. The zeta potential reflected the PCL and polysorbate 80 surface characteristics (−14.3±1.3). All formulations showed a slightly acidic profile (pH: 4.9 ± 0.3). Chromatographic technique indicated that drug content was close to 100%, considering the theoretical value (0.8 mg/ml). NC-TAC showed an encapsulation efficiency close to 100%.

3.2. NC-TAC favors CD4 T cell regulatory profile in AIH

CD4 T cells isolated from the peripheral blood of healthy subjects and AIH patients were tested for their ability to proliferate in the presence of CD3/CD28 T cell activation and IL-2 and in the presence of NC, NC-TAC, TAC or vehicle. Proliferation of CD4 T cells was decreased following treatment with NC-TAC in both healthy subjects and AIH patients (Fig. 1A-B), although this decrease reached statistical significance only in the latter (Fig. 1B). Treatment with TAC reduced CD4 T cell proliferation in both groups (Fig. 1A-B). Within each group, no differences were noted between the effects of NC-TAC and TAC on CD4 T cell proliferative response (Fig. 1A-B).

Figure 1. Effects of NC-TAC on CD4 T cell proliferation and apoptosis.

CD4 T cells were isolated from the peripheral blood of AIH patients and healthy subjects (HS) and tested for their ability to proliferate in the presence of Dynabeads Human T activator CD3/CD28, IL-2 and vehicle, empty nanocapsules (NC), tacrolimus (TAC)-loaded NC (NC-TAC) or unencapsulated TAC. Cell proliferation was assessed using CellTiter 96 Aqueous One Solution Cell Proliferation Assay and measured as absorbance at 490 nm. (A-B) Violin plots showing the absorbance at 490 nm of CD4 T cells in the presence of vehicle, NC, NC-TAC or TAC (HS, n=5; AIH, n=7). Early and late apoptotic CD4 T cells were detected as Annexin V⁺ (early apoptotic) and Annexin V⁺/Propidium Iodide⁺ (late apoptotic) lymphocytes by flow cytometry. Violin plots representing the frequency of (C-D) early apoptotic and (E-F) late apoptotic CD4 T cells in the presence of vehicle, NC, NC-TAC or TAC (HS, n=5; AIH, n=5). *P≤0.05; **P≤0.01; ***P≤0.001 (one-way ANOVA test followed by Tukey’s multiple comparisons test). In each violin plot, median and quartiles are shown. In AIH, addition of NC-TAC controls the proliferation of CD4 T lymphocytes, leaving the frequency of apoptotic cells substantially unchanged.

When analyzing apoptosis, a decrease in the proportion of early apoptotic cells (i.e., Annexin-V⁺ and PI⁻) (Supplemental Fig. 1) was noted in CD4 T lymphocytes of healthy subjects following exposure to NC, NC-TAC or TAC when compared to vehicle-treated cells (Fig. 1C); a decrease in the frequency of early apoptotic cells was also noted in CD4 T lymphocytes of AIH patients following exposure to NC (Fig. 1D). The proportion of early apoptotic cells was higher in the presence of TAC than NC or NC-TAC in healthy subjects (Fig. 1C) and tended to be higher than in the presence of NC in CD4 T cells isolated from AIH patients (Fig. 1D). In healthy subjects, an increase in the frequency of late apoptotic cells (Annexin-V⁺ and PI⁺) was noted in CD4 T lymphocytes exposed to TAC when compared to NC, NC-TAC or vehicle (Fig. 1E). In AIH, no change in the proportion of late apoptotic cells was noted following exposure of CD4 T lymphocytes to NC, NC-TAC or TAC (Fig. 1F).

We then tested the effects of NC, NC-TAC or TAC on the phenotype of CD4 T cells (Fig. 2A-E, Supplemental Fig. 2). In AIH samples, addition of NC-TAC resulted in increased proportions of CD4⁺FOXP3⁺ cells when compared to TAC (Fig. 2A), and higher frequencies of CD4⁺IL-10⁺ cells when compared to vehicle (Fig. 2C). Exposure to TAC resulted in lower frequencies of CD4⁺FOXP3⁺ cells when compared to NC (Fig. 2A) and tended to increase the frequency of CD4⁺IL-17A⁺ cells when compared to NC-TAC or NC (Fig. 2D). In healthy subject samples, addition of TAC increased the frequency of CD4⁺RORC⁺ (Fig. 2B) and CD4⁺IFNγ⁺ (Fig. 2E) when compared to vehicle or NC, and the percentage of CD4⁺IL-17A⁺ cells (Fig. 2D) when compared to NC-TAC. A summary of the effects of NC-TAC and TAC on CD4 T cell phenotype is provided in Supplemental Table 3.

Figure 2. NC-TAC favors CD4 T cell regulatory phenotype in AIH.

The frequency of CD4 T cells positive for FOXP3, RORC, IL-10, IL-17A and IFNγ was measured following treatment with NC, NC-TAC, TAC or vehicle by flow cytometry. Violin plots representing the frequency of (A) CD4⁺FOXP3⁺, (B) CD4⁺RORC⁺, (C) CD4⁺IL-10⁺, (D) CD4⁺IL-17A⁺ and (E) CD4⁺IFNγ⁺ cells following exposure to vehicle, NC, NC-TAC or TAC (HS, n=6-8; AIH, n=6-12). *P≤0.05; **P≤0.01 (one-way ANOVA test followed by Tukey’s multiple comparisons test). In each violin plot, median and quartiles are shown. NC-TAC imparts a regulatory phenotype to CD4 T cells of AIH patients.

We next considered differences in the response to NC, NC-TAC or TAC between CD4 cells obtained from AIH patients at remission and during relapse. In AIH patients at remission, we noted higher frequencies of CD4⁺FOXP3⁺ cells in the presence of NC-TAC when compared to TAC (Supplemental Fig. 4A) and lower frequencies of CD4⁺FOXP3⁺ cells in the presence of TAC when compared to NC (Supplemental Fig. 4A). In AIH patients at relapse, we noted higher proportions of CD4⁺IL-17A⁺ cells in the presence of TAC (Supplemental Fig. 4D). No differences in the proportion of RORC⁺, IL-10⁺ and IFNγ⁺ lymphocytes were noted within CD4 cells obtained from AIH patients at remission and relapse in response to NC, NC-TAC or TAC (Supplemental Fig. 4B-C and 4E).

Overall, these data indicate that NC-TAC controls the proliferation of and imparts regulatory features to CD4 T lymphocytes of AIH patients, whereas unencapsulated TAC might favor a pro-inflammatory phenotype.

3.3. NC-TAC ameliorates suppressive function and preserves immunophenotype of AIH Tregs

Given the beneficial effects of NC-TAC on the CD4 T cell compartment, we tested the impact of these nanoformulations on the proliferative response of Tregs isolated from the peripheral blood of healthy subjects and AIH patients. When compared to vehicle or NC, NC-TAC did not change the proliferation of healthy subject and AIH Tregs (Fig. 3A). In healthy subjects, addition of TAC led to decreased Treg cell proliferation when compared to vehicle or NC (Fig. 3A).

Figure 3. NC-TAC enhances Treg suppressive function in AIH.

Treg cells, isolated from the peripheral blood of HS and AIH patients were tested for their ability to proliferate in the presence of Dynabeads Human T activator CD3/CD28 and IL-2, following exposure to vehicle, NC, NC-TAC or unencapsulated TAC. (A) Violin plots showing the absorbance at 490 nm of Tregs in the presence of vehicle, NC, NC-TAC or TAC (HS, n=16; AIH, n=9). Treg suppression was measured as percentage inhibition of CD4⁺CD25⁻ cell proliferation. (B) Violin plots representing the percentage inhibition of CD4⁺CD25⁻ cell proliferation following co-culture with Tregs exposed to vehicle, NC, NC-TAC or TAC (HS, n=6; AIH, n=5). *P≤0.05; **P≤0.01 (one-way ANOVA test followed by Tukey’s multiple comparisons test). In each violin plot, median and quartiles are shown. Exposure to NC-TAC enhances AIH Treg suppressive function.

We next evaluated the effects of NC-TAC on Treg suppressive function, assessed as the ability to control CD4⁺CD25⁻ cell proliferation. In AIH, exposure to NC-TAC boosted Treg suppression when compared to vehicle or NC (Fig. 3B). No difference in suppressive function was noted between Tregs obtained from AIH patients at remission and during relapse following cell exposure to NC, NC-TAC or TAC (Supplemental Fig. 5).

As we have previously shown that AIH Tregs acquire features of T effectors in an inflammatory milieu [31], we tested the effects of NC-TAC on Tregs exposed to a pro-inflammatory challenge consisting of IL-6 and IL-1β (Fig. 4A-D). Due to the high cell yield required for this experiment, Tregs were obtained following polarization of CD4 naïve lymphocytes in the presence of IL-2, TGF-β and Dynabeads Human T activator CD3/CD28. The phenotype of Tregs polarized from CD4 naïve cells was comparable to that of freshly isolated Tregs, as we have previously reported [26, 32]. In AIH, exposure of Tregs to pro-inflammatory challenge tended to decrease FOXP3 and increase IL-17A levels (Fig. 4A, D). Addition of NC-TAC abrogated these effects by boosting FOXP3 and decreasing IL-17A (Fig 4A, D). Levels of FOXP3 and IL-17A were respectively higher and lower in NC-TAC when compared to vehicle-treated Tregs in the absence of pro-inflammatory challenge (Fig. 4A, D). In AIH, addition of NC-TAC boosted Treg IL-10 levels when compared to vehicle in the absence or presence of pro-inflammatory challenge (Fig. 4C). Addition of TAC increased Treg IL-10 levels when compared to vehicle in the absence of IL-6 and IL-1β in both healthy subjects and AIH patients; and when compared to vehicle in the presence of pro-inflammatory challenge in AIH patients (Fig. 4C). Levels of RORC were not impacted upon addition of NC-TAC, NC or TAC in both healthy subject and AIH Tregs (Fig. 4B). In Tregs obtained from AIH patients at remission, an increase in FOXP3 mRNA levels was noted following exposure to NC-TAC when compared to TAC or vehicle, in the presence of proinflammatory challenge (Supplemental Fig. 6A). A decrease in IL-17A levels was also noted in Tregs of AIH patients at remission upon exposure to NC-TAC or TAC when compared to vehicle, in the absence or presence of pro-inflammatory challenge (Supplemental Fig. 6D). No differences in the levels of RORC and IL-10 were noted between Tregs obtained from AIH patients at remission and at relapse in response to NC, NC-TAC or TAC (Supplemental Fig. 6B-C).

Figure 4. NC-TAC maintains AIH Treg phenotype in an inflammatory milieu.

The preservation of Treg phenotype was tested following exposure to an inflammatory challenge consisting of IL-6 and IL-1β. Due to the high cell yield required for this experiment, Tregs were obtained from CD4 naïve cells exposed to IL-2, TGF-β and Dynabeads Human T activator CD3/CD28 for T cell expansion. Violin plots of (A) FOXP3, (B) RORC, (C) IL-10 and (D) IL-17A mRNA levels in Tregs exposed to vehicle in the absence or presence of pro-inflammatory challenge, and following treatment with NC, NC-TAC or TAC and pro-inflammatory challenge (HS, n=9; AIH, n=10). Results are presented as fold change compared to vehicle in the absence of pro-inflammatory challenge. *P≤0.05; **P≤0.01 (one-way ANOVA test followed by Tukey’s multiple comparisons test). In each violin plot, median and quartiles are shown. Treatment with NC-TAC preserves the phenotype of AIH Tregs in the presence of a pro-inflammatory challenge.

A summary of the effects of NC-TAC and TAC on Treg cell suppressive function and immunophenotype in the presence of challenge is provided in Supplemental Table 3.

Overall, these data show that NC-TAC boosts AIH Treg suppression and maintains Treg immunophenotype in a pro-inflammatory environment.

3.4. Treatment with NC-TAC controls liver inflammation in vivo

The effects of NC-TAC were tested in vivo using a well-established model of T cell-mediated liver injury. In this model, liver injury was induced by administering Con-A to NOD/scid/gamma immunodeficient mice following reconstitution with CD4 T cells obtained from the peripheral blood of one healthy blood donor [25, 26]. After CD4 T cell reconstitution was verified by flow cytometry, mice were treated with NC-TAC, NC, TAC or vehicle, administered Con-A and sacrificed four hours later. Administration of NC-TAC resulted in decreased ALT levels when compared to vehicle (Fig. 5A) and in a decreased mHAI score and limited lymphoplasmacytic infiltration on histology when compared to vehicle, NC or TAC (Fig. 5B-C). When analyzing the phenotypic profile of intrahepatic human CD4 T cells (Fig. 5D-G and Supplemental Fig. 7), we noted increased frequencies of CD4⁺FOXP3⁺ cells in mice treated with NC-TAC (Fig. 5D) and decreased frequencies of CD4⁺IL-17A⁺ cells in mice treated with NC-TAC or TAC, when compared with mice treated with vehicle (Fig. 5F). No changes in the frequencies of intrahepatic CD4⁺IL-10⁺ (Fig. 5E) and CD4⁺IFNγ⁺ (Fig. 5G) cells were noted in mice treated with NC-TAC, NC or TAC. Treatment with NC-TAC, NC or TAC did not alter splenic histological architecture (Supplemental Fig. 7B and Supplemental Fig. 8A) and resulted in no changes in the frequency of splenic CD4⁺FOXP3⁺, CD4⁺IL-10⁺, CD4⁺IL-17A⁺ and CD4⁺IFNγ⁺ lymphocytes (Supplemental Fig. 8B-E). A summary of the effects of NC-TAC and TAC on clinical and immunological parameters in NOD/scid/gamma mice exposed to Con-A liver injury is provided in Supplemental Table 4.

Figure 5. NC-TAC ameliorates liver inflammation in vivo.

NOD/scid/gamma mice were reconstituted with human CD4 T cells and treated with vehicle, NC, NC-TAC or unencapsulated TAC twenty hours before exposure to acute hepatitis induced by Concanavalin-A (Con-A). Mice were sacrificed four hours after being exposed to Con-A. Mice reconstituted with human CD4 cells but unexposed to Con-A (no Con-A group) served as controls. Box and whiskers plots of (A) serum ALT levels and (B) the Ishak modified HAI (mHAI) score and the lymphoplasmacytic infiltration score in mice unexposed to Con-A (n=2) or treated with vehicle (n=5), NC (n=5), NC-TAC (n-=5) or TAC (n=5). (C) Hematoxylin and Eosin and human CD3 staining of liver sections (original magnification, ×10, scale bar: 200 μm), obtained from representative mice treated with vehicle, NC, NC-TAC or TAC. Box and whiskers plots representing the frequency of intra-hepatic CD4 T lymphocytes positive for (D) FOXP3, (E) IL-10, (F) IL-17A and (G) IFNγ. In all panels, the no Con-A group served as baseline reference only, not being powered for formal comparison. *P≤0.05; **P≤0.01 (one-way ANOVA test followed by Tukey’s multiple comparisons test).

Overall, these data indicate that NC-TAC controls liver inflammation in vivo while decreasing the proportion of effector CD4⁺IL-17A⁺ cells.

4. Discussion

Our study shows that NC-TAC has beneficial effects by favoring regulatory immune responses in vitro and by ameliorating T cell-mediated liver injury in vivo.

Our data indicate that the lipid-core nanocapsules achieved a uniform size distribution showing narrow dispersity and high drug encapsulation efficiency, which underscores their viability as drug delivery carriers [23]. Notably, the inclusion of TAC did not significantly affect particle size, thereby maintaining the nanoformulation stability and macroscopic homogeneity. The slightly negative zeta potential due to the polysorbate 80 coating acting by steric hindrance and the slightly acidic pH profile of the nanocapsules further contribute to their stability and physiological compatibility, enhancing their potential for biomedical application [23, 33].

Our findings indicate that compared to unencapsulated TAC, NC-TAC boosts CD4 cell regulatory properties in AIH samples, as reflected by heightened proportions of cells positive for FOXP3, the Treg transcription factor, and for IL-10, an anti-inflammatory cytokine that has been linked to Treg suppression [34, 35].

Furthermore, exposure to NC-TAC enhances Treg ability to suppress and preserves their phenotype in the presence of an inflammatory challenge consisting of IL-6 and IL-1β, two cytokines involved in the differentiation of human Th17 cells. In this set of experiments, exposure to NC-TAC reconstitutes FOXP3 and boosts IL-10 levels while controlling IL-17A levels. That NC-TAC not only enhances the suppressive function of Tregs but also maintains their phenotype is an important finding, especially in the context of autoimmune diseases, where Tregs tend to become plastic and acquire effector cell features. Enhancing the pool of Tregs has critical implications for the reconstitution of immunotolerance, as its breakdown is closely linked with numerical and functional Treg impairment [10]. The evidence that treatment with unencapsulated TAC induces an increase in the percentage of IFNγ⁺ and IL-17A⁺ CD4 T cells mirrors our previous findings showing an increase in the production of these pro-inflammatory cytokines by CD4 T lymphocytes after a forty-eight hour exposure to immunosuppressive drugs including TAC [36].

A direct comparison between the effects of NC-TAC and TAC on CD4 and Treg cells indicates that NC-TAC boosts the proportion of FOXP3⁺ while decreasing that of IL-17A⁺ cells within CD4 lymphocytes and increases FOXP3 levels in the presence of pro-inflammatory challenge. These effects are noted in AIH and, at least in part, in healthy subject-derived cells. These data further support the immunomodulatory potential of NC-TAC when compared to unencapsulated TAC.

Some of the immunomodulatory properties of NC-TAC (i.e., increased CD4⁺FOXP3⁺ cell frequencies, enhanced FOXP3 and reduced IL-17A levels in the presence of pro-inflammatory stimulus) appear to be present in cells of patients studied at remission, although studies with a larger number of patients per subgroup should be carried out to better characterize and validate these differences. Larger studies would be also needed to determine the effects of each patient’s individual treatment regimen on CD4 and Treg response to NC-TAC.

We should note that the in vitro data were obtained from CD4 and Treg cells isolated from AIH patients, none of whom were on TAC at the time of PBMC collection. As this might represent a limitation, future studies should also include patients on TAC treatment to evaluate the effects of NC-TAC in the context of prior or current ex vivo exposure to unencapsulated TAC.

Beneficial effects of NC-TAC have been also noted in vivo in a model of acute T cell-mediated liver injury induced by Con-A in NOD/scid/gamma mice reconstituted with human CD4 T cells. Treatment with NC-TAC resulted in decreased ALT levels and histological score - also when compared to free TAC - and in increased proportions of FOXP3⁺ cells among hepatic CD4 T lymphocytes. As a decrease in IL-17A producing CD4 T cells is noted in the presence of NC-TAC and unencapsulated TAC, it is likely that the beneficial effects of NC-TAC are predominantly linked to the increase in CD4⁺FOXP3⁺ lymphocytes. These findings further support the evidence that NC-TAC displays anti-inflammatory properties. Should NC-TAC be proven to enable a targeted TAC delivery in vivo, the translational implications would be significant. The favorable profile associated with enhanced Treg immunity makes these nanoformulations particularly suitable for AIH, potentially limiting inflammation while boosting Tregs and possibly favoring immunotolerance. Future studies are needed to examine the effects of NC-TAC on the function and activation status of other immune cells, i.e., B cells, macrophages and CD8 lymphocytes, which are also involved in AIH pathogenesis [37-40]. Additional investigations should evaluate the long-term effects of NC-TAC in AIH-specific pre-clinical models, obtained following transient overexpression of IL-12 in C57BL/6 mice [41] or upon immunization of human transgenic HLA-DR3 mice with a DNA plasmid coding for human liver autoantigens [42]. Both models represent valuable tools to recapitulate the chronic and relapsing-remitting nature of the disease [41, 42].

Overall, our study shows that NC-TAC displays immunomodulatory effects in vitro, which may contribute to the amelioration of T cell-mediated liver damage induced by Con-A in humanized mice. Although future work is needed to validate the effects on Treg functionality and to evaluate the kinetics and pharmacodynamic properties of these nanoformulations in vivo, the current findings support the potential of nanotechnology-based approaches for immune modulation in the context of autoimmune diseases like AIH. These approaches would open avenues for additional preclinical and clinical research aimed at optimizing and validating the applicability of this innovative therapeutic approach.

Highlights.

• NC-TAC imparts a regulatory phenotype to AIH-derived CD4 T cells

• NC-TAC enhances the function of AIH Tregs

• NC-TAC ameliorates Concanavalin-A-induced liver injury in humanized mice

• NC-TAC induces and maintains Treg immune responses in AIH

Abbreviations:

AIH

autoimmune hepatitis

PBMCs

peripheral blood mononuclear cells

Treg

regulatory T cell

con-A

concanavalin-A

TAC

tacrolimus

NC

nanocapsules

NC-TAC

tacrolimus loaded nanocapsules