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Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity.

ABSTRACT: Individuals homozygous for the "Z" mutation in alpha-1 antitrypsin deficiency are known to be at increased risk for liver disease. It has also become clear that some degree of risk is similarly conferred by the heterozygous state. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of a single Z alpha-1 antitrypsin (ZAAT) allele on hepatocyte biology. Here, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-hepatocytes (iHeps). We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations including altered endoplasmic reticulum (ER) and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in cell subpopulations. Our model of MZ heterozygosity thus provides evidence that a single Z allele is sufficient to disrupt hepatocyte homeostatic function.

SUBMITTER: Kaserman JE

PROVIDER: S-EPMC9780780 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity.

Kaserman Joseph E JE Werder Rhiannon B RB Wang Feiya F Matte Taylor T Higgins Michelle I MI Dodge Mark M Lindstrom-Vautrin Jonathan J Bawa Pushpinder P Hinds Anne A Bullitt Esther E Caballero Ignacio S IS Shi Xu X Gerszten Robert E RE Brunetti-Pierri Nicola N Liesa Marc M Villacorta-Martin Carlos C Hollenberg Anthony N AN Kotton Darrell N DN Wilson Andrew A AA

Cell reports 20221201 10

Individuals homozygous for the "Z" mutation in alpha-1 antitrypsin deficiency are known to be at increased risk for liver disease. It has also become clear that some degree of risk is similarly conferred by the heterozygous state. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of a single Z alpha-1 antitrypsin (ZAAT) allele on hepatocyte biology. Here, we describe the derivation of syngeneic induced pluripotent stem cells ...[more]

PMID: 36476855

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Project description:The serpin family A member 1 (SERPINA1) Z allele is present in approximately one in 25 individuals of European ancestry. Z allele homozygosity (Pi*ZZ) is the most common cause of alpha 1-antitrypsin deficiency and is a proven risk factor for cirrhosis. We examined whether heterozygous Z allele (Pi*Z) carriers in United Kingdom (UK) Biobank, a population-based cohort, are at increased risk of liver disease. We replicated findings in Massachusetts General Brigham Biobank, a hospital-based cohort. We also examined variants associated with liver disease and assessed for gene-gene and gene-environment interactions. In UK Biobank, we identified 1,493 cases of cirrhosis, 12,603 Z allele heterozygotes, and 129 Z allele homozygotes among 312,671 unrelated white British participants. Heterozygous carriage of the Z allele was associated with cirrhosis compared to noncarriage (odds ratio [OR], 1.53; P = 1.1×10-04); homozygosity of the Z allele also increased the risk of cirrhosis (OR, 11.8; P = 1.8 × 10-09). The OR for cirrhosis of the Z allele was comparable to that of well-established genetic variants, including patatin-like phospholipase domain containing 3 (PNPLA3) I148M (OR, 1.48; P = 1.1 × 10-22) and transmembrane 6 superfamily member 2 (TM6SF2) E167K (OR, 1.34; P = 2.6 × 10-06). In heterozygotes compared to noncarriers, the Z allele was associated with higher alanine aminotransferase (ALT; P = = 4.6 × 10-46), aspartate aminotransferase (AST; P = 2.2 × 10-27), alkaline phosphatase (P = 3.3 × 10-43), gamma-glutamyltransferase (P = 1.2 × 10-05), and total bilirubin (P = 6.4 × 10-06); Z allele homozygotes had even greater elevations in liver biochemistries. Body mass index (BMI) amplified the association of the Z allele for ALT (P interaction = 0.021) and AST (P interaction = 0.0040), suggesting a gene-environment interaction. Finally, we demonstrated genetic interactions between variants in PNPLA3, TM6SF2, and hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13); there was no evidence of epistasis between the Z allele and these variants. Conclusion: SERPINA1 Z allele heterozygosity is an important risk factor for liver disease; this risk is amplified by increasing BMI.

Project description:For patients with non-cirrhotic liver-based metabolic disorders, hepatocyte transplantation can be an effective treatment. However, long-term function of transplanted hepatocytes following infusion has not been achieved due to insufficient numbers of hepatocytes reaching the liver cell plates caused by activation of the instant blood-mediated inflammatory reaction (IBMIR). Our aim was to determine if the natural immune modulator, alpha-1 antitrypsin (AAT), could improve engraftment of transplanted hepatocytes and investigate its mechanism of action. A tubing loop model was used to analyse activation of the IBMIR when human hepatocytes were in contact with ABO-matched blood and 4 mg/ml AAT. Platelet and white cell counts, complement and cytokine expression were analysed. To determine if AAT could improve short-term engraftment, female rats underwent tail vein injection of AAT (120 mg/kg) or water (control) prior to the intrasplenic transplantation of 2 × 107 male hepatocytes. At 48 h and 1 week, livers were collected for analysis. In our loop model, human hepatocytes elicited a significant drop in platelet count with thrombus formation compared to controls. Loops containing AAT and hepatocytes showed no platelet consumption and no thrombus formation. Further, AAT treatment resulted in reduced IL-1β, IL-6 and IFN-γ and increased IL-1RA compared to untreated loops. In vivo, AAT significantly improved engraftment of rat hepatocytes compared to untreated at 48 h. AAT infusion may inhibit the IBMIR, thus improving short-term engraftment of donor hepatocytes and potentially improve the outcomes for patients with liver-based metabolic disease. KEY MESSAGES: • Alpha-1 antitrypsin (AAT) acts as an immune modulator to improve the efficacy of hepatocyte transplantation. • Treatment with AAT decreased thrombus formation and pro-inflammatory cytokine expression in a tubing loop model. • AAT significantly improved engraftment of donor hepatocytes within the first 48 h post transplantation.

Project description:Background & aimsA single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention.MethodsOur functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease.ResultsWe report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease.ConclusionsPDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD.Lay summaryProtein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.

Dataset Information

Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity.

Publications

Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity.

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