Comparison of wild-type and high-risk PNPLA3 variants in a human biomimetic liver microphysiology system for metabolic dysfunction-associated steatotic liver disease precision therapy

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide health epidemic with a global occurrence of approximately 30%. The pathogenesis of MASLD is a complex, multisystem disorder driven by multiple factors, including genetics, lifestyle, and the environment. Patient heterogeneity presents challenges in developing MASLD therapeutics, creating patient cohorts for clinical trials, and optimizing therapeutic strategies for specific patient cohorts. Implementing pre-clinical experimental models for drug development creates a significant challenge as simple in vitro systems and animal models do not fully recapitulate critical steps in the pathogenesis and the complexity of MASLD progression. To address this, we implemented a precision medicine strategy that couples the use of our liver acinus microphysiology system (LAMPS) constructed with patient-derived primary cells. We investigated the MASLD-associated genetic variant patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 (I148M variant) in primary hepatocytes as it is associated with MASLD progression. We constructed the LAMPS with genotyped wild-type and variant PNPLA3 hepatocytes, together with key non-parenchymal cells, and quantified the reproducibility of the model. We altered media components to mimic blood chemistries, including insulin, glucose, free fatty acids, and immune-activating molecules to reflect normal fasting (NF), early metabolic syndrome (EMS), and late metabolic syndrome (LMS) conditions. Finally, we investigated the response to treatment with resmetirom, an approved drug for metabolic syndrome-associated steatohepatitis (MASH), the progressive form of MASLD. This study, using primary cells, serves as a benchmark for studies using "patient biomimetic twins" constructed with patient induced pluripotent stem cell (iPSC)-derived liver cells using a panel of reproducible metrics. We observed increased steatosis, immune activation, stellate cell activation, and secretion of pro-fibrotic markers in the PNPLA3 GG variant compared to the wild-type CC LAMPS, consistent with the clinical characterization of this variant. We also observed greater resmetirom efficacy in the PNPLA3 wild-type CC LAMPS compared to the GG variant in multiple MASLD metrics, including steatosis, stellate cell activation, and the secretion of pro-fibrotic markers. In conclusion, our study demonstrates the capability of the LAMPS platform for the development of MASLD precision therapeutics, enrichment of patient cohorts for clinical trials, and optimization of therapeutic strategies for patient subgroups with different clinical traits and disease stages.

Keywords: metabolic dysfunction-associated steatotic liver disease; microphysiology systems; patatin-like phospholipase domain-containing protein 3; precision medicine; reproducibility.

FIGURE 1

Overview of the precision medicine approach used to investigate genotype-specific MASLD progression and drug response using PNPLA3 genotyped patient-derived cells in the liver acinus microphysiology system (LAMPS). (A) MASLD progression is complex due to multiple factors, including genetics, environment, and lifestyle, resulting in patient heterogeneity. The PNPLA3 rs738409 GG variant is highly associated with MASLD susceptibility (Romeo et al., 2008; Sookoian and Pirola, 2011; Rotman et al., 2010; Xu et al., 2015). To study this high-risk variant, we started with the genotyped PNPLA3 rs738409 GG variant and CC wild-type primary hepatocytes to use as a benchmark for studies using iPSC-derived liver cells (de I’Hortet et al., 2019; Takeishi et al., 2020). These genotyped primary hepatocytes were cultured with nonparenchymal cells (NPCs) including liver sinusoidal endothelial cells (LSECs), stellate cells, and Kupffer cells in the LAMPS (Lefever et al., 2022; Saydmohammed et al., 2021; Vernetti et al., 2016). (B) MASLD progression in the LAMPS was driven with specific medium formulations to mimic lifestyle effects, as previously described (Saydmohammed et al., 2021), and monitored through various clinically relevant metrics, including steatosis, stellate cell activation, inflammation, and fibrosis. Additionally, the efficacy of recently FDA-approved resmetirom (Rezdiffra™) (Harrison et al., 2024; Harrison et al., 2019) was assessed in the LAMPS, focusing on the early stage of MASLD progression using the EMS medium. (C) Results from these studies can then inform a precision medicine strategy that implements the use of patient-derived iPSCs to generate patient biomimetic twins (PBTs) for drug testing and development, as well as clinical trial patient cohort selection (Gough et al., 2021).

FIGURE 2

Increased steatosis in the PNPLA3 rs738409 GG variant LAMPS compared to the CC wild-type LAMPS is consistent with the clinical characterization of the GG variant. (A) Representative images of the LipidTOX-labeled PNPLA3 LAMPS maintained in NF, EMS, and LMS media. Magnification: 40×; scale is 50 µm. (B) Steatosis was quantified by quantitative fluorescence imaging of LipidTOX-labeled samples under each medium condition. Significant increases in steatosis were observed in the PNPLA3 GG variant LAMPS compared to the CC wild-type LAMPS under all three medium conditions, consistent with the clinical characterization of this high-risk variant (Romeo et al., 2008). Data were obtained on day 8 with a minimum of n = 3 LAMPSs from each patient lot for each condition and plotted as the mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s test. p-value <0.05 was considered statistically significant. Statistical comparisons across medium conditions within each genotype were also performed, as shown in Supplementary Figure S5.

FIGURE 3

Increased pro-inflammatory cytokine production is observed in the PNPLA3 rs738409 GG variant compared to the CC wild-type LAMPS, demonstrating enhanced immune activation. (A–C: absolute cytokine values) For NF medium, a significant increase in IL-8 secretion (C) was observed in the PNPLA3 GG variant compared to the CC wild-type LAMPS. For the LMS medium, significant increases in the secretion of both IL-6 (B) and IL-8 (C) were observed in the PNPLA3 GG variant compared to the CC wild-type LAMPS, consistent with studies demonstrating increased immune activation and inflammation associated with the PNPLA3 GG variant (Kabbani et al., 2022; Krawczyk et al., 2020; Kirchmeyer et al., 2023). No significant differences in CCL2 secretion were observed between the PNPLA3 GG variant and CC wild-type LAMPS in any medium condition. (D–F: normalized to NF values) Absolute cytokine values shown in A–C were normalized to their respective NF control for each study. For normalized cytokine values, while no significant differences between the PNPLA3 GG and CC LAMPSs were observed in either EMS or LMS medium, the overall secretion levels of CCL2, IL-6, and IL-8 were all increased in EMS and LMS media compared to the NF medium. The statistical comparisons between medium types within each PNPLA3 genotype are shown in Supplementary Figure S7. Data were obtained on day 8 with a minimum of n = 3 LAMPSs from each patient ID for each medium condition. Plotted is the mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s test. p-value <0.05 was considered statistically significant.

FIGURE 4

Increased stellate cell activation and COL1A1 secretion are observed in the PNPLA3 GG variant LAMPS compared to the CC wild-type LAMPS, demonstrating a more severe pro-fibrotic phenotype, consistent with the clinical characterization of the GG variant. (A) Representative images of the αSMA-labeled PNPLA3-LAMPS maintained in NF, EMS, and LMS media. Magnification: 40×; scale: 50 µm. (B) A significant increase in αSMA integrated intensity was observed in the PNPLA3 GG variant LAMPS compared to the CC wild-type LAMPS in both NF and LMS media, indicating an increase in stellate cell activation. (C) In addition, a significant increase in COL1A1 secretion was also observed in the PNPLA3 GG variant compared to the CC wild-type LAMPS in both NF and LMS media. Taken together, these results demonstrate that the PNPLA3 variant is associated with increased stellate cell activation and subsequent secretion of pro-fibrotic markers (COL1A1), consistent with the clinical characterization of this high-risk variant (Valenti et al., 2010; Krawczyk et al., 2020; Singal et al., 2014; Shen et al., 2015). Data were obtained on day 8 with a minimum of n = 3 LAMPSs from each patient lot for each condition and plotted as the mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s test. p-values <0.05 were considered statistically significant. Statistical analyses comparing across medium types for each PNPLA3 genotype were also performed and are shown in Supplementary Figure S8.

FIGURE 5

Resmetirom treatment resulted in a greater reduction in steatosis in the PNPLA3 CC wild-type LAMPS than that in the GG variant LAMPS, demonstrating genotype-specific inhibition of MASLD progression. PNPLA3 LAMPS models were maintained for 8 days in the EMS medium containing resmetirom (1 μM) or DMSO vehicle control. The secretion of the sex hormone-binding globulin (SHBG) was assessed to determine the on-target pharmacodynamic activity of resmetirom, and LipidTOX staining was quantified to determine the effect of resmetirom on steatosis. (A) Treatment with 1 µM resmetirom increased SHBG secretion in both PNPLA3 LAMPSs, demonstrating the on-target pharmacodynamic activity of resmetirom. However, the PNPLA3 CC wild-type LAMPS displayed significantly higher SHBG secretion than the GG variant LAMPS. (B) Resmetirom treatment resulted in a significantly greater reduction in steatosis in the PNPLA3 CC wild-type LAMPS (∼33%) compared to the PNPLA3 GG variant (∼20%). (C) Representative images of LipidTOX-labeled PNPLA3 LAMPS maintained in the EMS medium with and without 1 µM resmetirom. Magnification: 40×; scale: 50 µm. Data were obtained on day 8 with n = 3 LAMPSs from each patient lot for each condition and were plotted as the average % vehicle ± SEM. Statistical significance was assessed by ANOVA with Tukey’s test. p-values <0.05 were considered statistically significant. Statistical analysis comparing resmetirom treatment with the vehicle control within each PNPLA3 genotype was performed, as shown in Supplementary Figure S10.

FIGURE 6

Resmetirom treatment did not result in any significant changes in pro-inflammatory reduction between the PNPLA3 GG variant and CC wild-type LAMPS. No significant differences in the reduction (% of vehicle control) in IL-6 (A), CCL2 (B), or IL-8 (C) were observed between the PNPLA3 GG variant and CC wild-type LAMPS upon treatment with 1 µM resmetirom. Data (% of vehicle control) were obtained on day 8 with a minimum of n = 3 LAMPSs from each patient lot for each condition and are plotted as ± SEM. Statistical significance was assessed by ANOVA with Tukey’s test. p-values <0.05 were considered statistically significant.

FIGURE 7

Resmetirom treatment resulted in a greater reduction in stellate cell activation and COL1A1 secretion in the PNPLA3 CC wild-type LAMPS than that in the GG variant LAMPS. (A) Representative images of the αSMA-labeled PNPLA3 LAMPS maintained in the EMS medium with 1 µM resmetirom or vehicle control. Magnification: 40×; scale: 50 µm. (B,C) A significantly greater reduction in both αSMA integrated intensity (B; 50% vs 25%) and the secretion of COL1A1 (C; 35% vs 0%) was observed in the PNPLA3 CC wild-type LAMPS compared to the PNPLA3 GG variant LAMPS. Data were obtained on day 8 with n = 3 LAMPSs from each patient lot for each condition and were plotted as the average % vehicle ± SEM. Statistical significance was assessed by ANOVA with Tukey’s test. p-values <0.05 were considered statistically significant. Statistical analysis comparing resmetirom treatment with the vehicle control within each genotype was performed, as shown in Supplementary Figure S12.