Hepatocyte Transplantation Rebalances Cytokines for Hepatic Regeneration in Rats with Ataxia Telangiectasia Mutated Pathway-Related Acute Liver Failure

Fadi-Luc Jaber; Yogeshwar Sharma; Sanjeev Gupta

doi:10.1016/j.ajpath.2022.10.003

. 2023 Jan;193(1):27–38. doi: 10.1016/j.ajpath.2022.10.003

Hepatocyte Transplantation Rebalances Cytokines for Hepatic Regeneration in Rats with Ataxia Telangiectasia Mutated Pathway-Related Acute Liver Failure

Fadi-Luc Jaber ^∗,^†, Yogeshwar Sharma ^∗,^†, Sanjeev Gupta ^∗,^†,^‡,^§,^¶,^‖,^∗∗,^∗

PMCID: PMC9768683 PMID: 36309105

Abstract

Inadequate DNA damage response related to ataxia telangiectasia mutated gene restricts hepatic regeneration in acute liver failure. Resolving mechanistic gaps in liver damage and repair requires additional animal models that are unconstrained by ultrarapid and unpredictable mortalities or substantial divergences from human pathology. This study used Fischer 344 rats primed with the antitubercular drug, rifampicin, plus phenobarbitone, and monocrotaline, a DNA adduct-forming alkaloid. Rifampicin and monocrotaline can cause liver failure in people. This regimen resulted in hepatic oxidative stress, necrosis, DNA double-strand breaks, liver test abnormalities, altered serum cytokine expression, and mortality. Healthy donor hepatocytes were transplanted ectopically in the peritoneal cavity to study whether they could supply metabolic support and rebalance inflammatory or protective cytokines affecting liver regeneration events. Hepatocyte transplantation increased candidate cytokine levels (granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, IL-10, and IL-12), leading to Atm, Stat3, and Akt signaling in hepatocytes and nonparenchymal cells, lowering of inflammation, and improvements in intermediary metabolism, DNA repair, and hepatocyte proliferation. Such control of DNA damage and inflammation, along with stimulation of hepatic growth, offers paradigms for cell signaling to restore hepatic homeostasis and regeneration in acute liver failure. Further studies of molecular pathways of high pathobiological impact will advance the knowledge of liver regeneration.

The syndrome of acute liver failure (ALF) arises from multiple etiologies, including drug-induced liver injury, viruses, toxins, ischemia, immunologic disorders, and various other causes.¹ Common to these diverse etiologies in ALF is the sudden loss of liver parenchyma, resulting in hepatic insufficiencies, altered mental status, liver test abnormalities, susceptibility to intercurrent sepsis or multi-organ failure, and mortality due to impaired liver regeneration. However, the molecular pathology of ALF is complex and incompletely defined, although oxidative stress, mitochondrial dysfunction, inadequate DNA damage response (DDR), inflammation, and yet other events may lead to hepatic growth arrest.2, 3, 4 Molecular studies, including the recent single-cell or spatiotemporally visualized transcriptomics in animal injury models for acetaminophen (APAP), chemicals, or ischemia, re-emphasize that deleterious signals affecting liver regeneration and inflammation can arise not only from hepatocytes themselves, but also from adjacent nonparenchymal liver cells (NPCs),⁵^,⁶ infiltrating inflammatory cells,⁷ or even the distant gut microbiome.⁵^,⁸

More importantly, healthy hepatocytes and other cell types, such as liver sinusoidal endothelial cells (LSECs), can secrete numerous cytokines, growth factors, receptors, and extracellular matrix components, including those with major consequences for liver regeneration.²^,⁹^,¹⁰ During ALF in humans, and animals, inflammatory and trophic factors are imbalanced,²^,¹¹ in part, because damaged hepatocytes begin to secrete inflammatory cytokines more and trophic cytokines less.² This secretory disruption in damaged hepatocytes arises from intracellular perturbations. For instance, ataxia telangiectasia mutated (ATM) gene is a major determinant of liver cell homeostasis.¹²^,¹³ The ATM gene through downstream pathway members is a primary determinant for cell death, survival, and growth fate decisions, including regulation of DDR, NF-κB–modulated inflammation, and other critical processes.¹²^,14, 15, 16 Consequently, the onset of ATM insufficiency in ALF leads to liver growth arrest and failure of liver regeneration.²^,³^,¹⁵^,¹⁷^,¹⁸ Thus, this pathway is critical for modeling the pathophysiology and therapeutics of ALF.

Although orthotopic liver transplantation can rescue ALF, it is mired with donor organ shortages, perioperative complications, and necessity for life-long immunosuppression. This makes alternatives to overcome liver growth arrest such as cell or tissue therapies more attractive.²^,¹⁹ These alternatives offer metabolic support for aiding survival and aid the release of regenerative factors in the damaged liver, as in the healthy hepatocytes, but not LSECs.² Remarkably, severe hepatic injury, as studied in cell or mouse models of APAP toxicity, can profoundly alter liver gene expression [eg, new expression of granulocyte colony-stimulating factor (G-CSF) receptor].²^,¹⁷ The G-CSF released by transplanted cells or administered exogenously can activate Janus kinase (JAK)/STAT3 signaling, which, in turn, can restore ATM pathway.²^,³^,¹⁷ This aspect of soluble factor replacement needs further testing in ALF models. Although numerous animal models for ALF involving drugs, toxins, transgenes, ischemia, surgical resection, to name a few, are available,⁵^,¹⁹^,²⁰ these may not recapitulate critical aspects of hepatic DDR, inflammation, or growth arrest. Confounding from ultrarapid mortality or spontaneous recovery due to individual or strain-level differences in animal biology, cofactors, or other events related to drug-induced liver injury may be reflected even in cell line models.²¹^,²²

Rifampicin (Rif) and phenytoin (Phen), which are used for tuberculosis and epilepsy, respectively, and monocrotaline (MCT), a DNA adduct forming plant-derived pyrrolizidine alkaloid, produce ATM insufficiency to cause ALF in immunodeficient nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice in C3H/He background, but with far less liver damage in mice from C57BL/6 background.²³^,²⁴ The Rif/Phen/MCT-induced ALF in NOD/SCID mice causes mortalities over several days, thus differing from LD₅₀ amounts of APAP with overnight mortality in one-half but little or no liver damage in the remainder.² This is significant for human health because Rif and Phen can cause ALF,²⁵ whereas inadvertent exposure to MCT can cause endothelial damage leading to hepatic sinusoidal obstruction syndrome, and also ALF.²⁵^,²⁶ Rif and Phen induce hepatic Cyp3A4 enzyme expression to redirect MCT metabolism, leading to concurrent damage in hepatocytes and LSECs.²³^,²⁴ The endothelial toxicity in rodents from MCT alone has been of interest for modeling pulmonary hypertension,²⁷ and for evaluating liver repopulation from transplanted hepatocytes or LSECs.¹³^,²⁸^,²⁹ Significantly, liver regeneration can be induced following ectopic transplantation of healthy hepatocytes in peritoneal cavity in both APAP and Rif/Phen/MCT mouse models.²^,²³ These concepts of ATM insufficiency and soluble factor signaling will be strengthened by their validation in additional species.

Herein, the Rif/Phen/MCT model of ALF was transferred to immunocompetent Fischer 344 (F344) rats, although similar MCT hepatotoxicity and oxidative stress required substitution of phenobarbitone (PB) for Phen,³⁰ in conjunction with Rif.²³^,²⁴ The Rif/PB/MCT protocol reproduces ATM dysregulation, liver test abnormalities, liver necrosis, impaired DDR, growth arrest, and adverse outcomes. Moreover, as animals are rescued by ectopic transplantation of hepatocytes, the following principles could be examined: whether hepatocyte transplantation would identify correction of imbalances in inflammatory or trophic cytokines, and whether ATM-relevant intracellular signaling activated by corrected cytokines and growth factors would be identifiable in hepatocytes and NPCs during liver regeneration.

Materials and Methods

Drugs and Reagents

Drugs and reagents were from Sigma-Aldrich Chemicals Co (St. Louis, MO). Rif was solubilized in 20 mol/L NaOH and PB or MCT in normal saline to pH 7.4.

Animals

The Animal Care and Use Committee at Albert Einstein College of Medicine (Bronx, NY) approved protocols according to NIH³¹ and other regulators. The F344 rats of 8 to 10 weeks age and weighing 150 to 200 g were from Charles River Laboratories (Kingston, NY). Animals housing used 14/10-hour light/dark cycles with unrestricted access to water and pelleted chow.

For ALF, i.p. 75 mg/kg Rif and 5 mg/kg PB were given once daily for 3 days, followed by i.p. 200 mg/kg MCT once on day 4.

For cell transplantation, isolation of donor hepatocytes from healthy F344 rats used two-step collagenase perfusion, as previously described.³⁰ Trypan blue dye exclusion showed cell viability exceeded 80%. Keeping cells on ice at 4°C maintained viability and cell transplantation intraperitoneally by 20-gauge needles was within 2 hours from their isolation. Each animal received 5 × 10⁷ hepatocytes suspended in serum-free RPMI 1640 culture medium (Gibco, Grand Island, NY) containing 1-mL Cytodex³ microcarriers (Amersham Biosciences, Piscataway, NJ). These 5 × 10⁷ cells approached 10% to 15% of the expected hepatocyte mass in healthy rat liver (3 × 10⁸ hepatocytes should represent 60% of 5 × 10⁸ total liver cells anticipated in 200-g rats). Use of 5 × 10⁷ cells followed previous dose-titration studies in mice showing rescue after i.p. transplantation of 10-fold fewer, body size–adjusted 5 × 10⁶ mouse or rat hepatocytes in APAP- or Rif/Phen/MCT-induced ALF.²^,²³ We transplanted cells 20 to 24 hours after completion of MCT dosing for ALF induction. Sham-treated animals serving as controls for cell transplantation studies received only vehicle and microcarriers.

Histology and Tissue Studies

Tissue samples were frozen in methylbutane to −80°C. Cryosections (5 μm thick) were fixed in ice-cold ethanol for hematoxylin and eosin staining or for histochemical localization of glycogen, glucose-6-phosphatase (G6P), and gamma glutamyltranspeptidase (GGT), as previously described.⁷^,³² Myeloperoxidase activity in polymorphonuclear neutrophils (PMNs) was stained with a kit (Sigma), as previously described.⁷ For immunostainings, sections were fixed in 4% paraformaldehyde in phosphate-buffered saline for 10 minutes, permeabilized in 0.3% Triton-X in phosphate-buffered saline for 15 minutes, and blocked in 3% goat serum in phosphate-buffered saline for 1 hour at room temperature. For nitrotyrosine and γ-H2A histone family member X (γH2AX), sections were incubated with rabbit anti-nitrotyrosine (1:200; A-21285; Invitrogen, Carlsbad, CA) or rabbit anti-γH2AX (1:100; 2595; Cell Signaling Technologies, Beverly, MA) in phosphate-buffered saline overnight at room temperature, and detected by goat anti-rabbit peroxidase-conjugated IgG (1:200; Sigma) with DAB+ (K3467; DakoCytomation, Glostrup, Denmark) and hematoxylin counterstain. Primary antibodies included rabbit Ki-67 (1:100; RB-9043; Thermo Fisher Scientific, Fremont, CA), rabbit phosphorylated STAT3 (pSTAT3; 1:100; 9145; Cell Signaling Technologies), rabbit phosphorylated ATM (1:100; MA1-46069; Thermo Fisher, Waltham, MA), or mouse monoclonal HSA-9 albumin antibody (1:100; A2672; Sigma), with overnight incubations at 4°C and visualization with Alexa-488 or Alexa-677 IgG conjugates (1:200; Cell Signaling Technologies) for 1 hour at room temperature. Nuclei were counterstained by Hoechst 33,342 dye (1:1000; H3570; Invitrogen) for 1 hour at room temperature.

Tissue Analysis

Morphometry used multiple samples per animal. Necrotic areas in tissues were measured under ×40 magnification by ImageJ software version 1.53 m (NIH, Bethesda, MD; https://imagej.nih.gov/ij/index.html, last accessed September 28, 2021). Quantitation of other events was in 20 to 30 high-power fields (×200 magnification) with the Cytation instrument (BioTek, Winoosky, VT).

Transcriptional Analysis

Total RNA was isolated from liver samples in healthy control rats and rats in ALF with or without cell therapy (n = 3 each) by TRIzol Reagent (Life Sciences, Carlsbad, CA). RNAs (1 μg) were converted to cDNAs by RT² PCR Array First Strand Kit (SA Biosciences, Frederick, MD). Rat Stress & Toxicity PathwayFinder RT² Profiler PCR Arrays (PARN-003Z; Qiagen, Germantown, MD) were used for 84 pathway genes, 5 housekeeping genes, 1 genomic DNA contamination control, 3 room temperature controls for cDNA conversion, and 3 other positive controls, according to manufacturer. Genes included DNA damage (n = 16), inflammation (n = 9), oxidative stress (n = 10), hypoxia (n = 11), cell death (n = 20), unfolded protein response (n = 12), or osmotic stress (n = 12), with overlaps in cases (Supplemental Table S1). Fold expression differences used 2-^ΔΔC_T method, as previously described.⁷ Housekeeping genes were used for separately normalizing experimental and control samples. Differences of ≥1.5-fold up or down were annotated for canonical pathways and upstream regulator networks in December 2021, using built-in tools by Ingenuity Pathway Analysis (IPA) software (winter 2021 release; Flintstone Knowledge Base; Qiagen). The prioritization of network analysis used –log (P values) and false discovery rate of <5% by Fisher exact test. Characterization of upstream regulator networks included z-scores, total regulator nodes, and P values for interactions in downstream pathway regulators.

Prothrombin Time

Blood samples were collected in 0.11-mol/L sodium citrate, as previously described.²³ Plasma was separated and diluted in water to 0.1-mL aliquots (7:3, v/v), preincubated at 37°C for 5 minutes, and incubated with 0.2-mL thromboplastin in water (Plastinex; 101,158; Bio/Data Corp., Horsham, PA). The time to clot formation was then measured.

Serologic Assays

Sera were stored at −80°C. Alanine aminotransferase and total bilirubin were assayed in clinical pathology laboratory. Kits to measure rat G-CSF to picograms per mL sensitivity (LS-F23183; LSBio, Seattle, WA), and allowing comparison of granulocyte-macrophage colony-stimulating factor (GM-CSF), ILs (IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12, and IL-13), interferon (IFN)-γ, and tumor necrosis factor (TNF) levels (MER-004A Multi-Analyte ELISArray; Qiagen), were used as recommended by the manufacturers. The serum G-CSF levels were used to normalize other cytokine levels in healthy rats.

Study Design

All studies used males and females in equal numbers. The animals were observed with or without cell transplantation for up to 14 days. Major findings were reproduced in experimental repeats. Samples of blood, liver, or i.p. conglomerates of transplanted cells and microcarriers were obtained at early (1 to 4 days) or late (10 to 14 days) times after Rif/PB/MCT regimen for mRNA expression, DDR, histology, and serologic parameters. Death was not an end point, although mortalities were documented. Moribund animals were euthanized and considered dead.

Statistical Analysis

The sample sizes were prospectively based on anticipated outcome differences of ≥30%. Data are shown as means ± SEM. The significances used t-test, χ² test, or analysis of variance with Bartlett post hoc test by GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA). For functional genomics, built-in IPA tools used Fisher exact test. P < 0.05 was considered significant. The findings were reproduced as appropriate in two to three replicate studies.

Results

The Rif/PB/MCT protocol was implemented using hepatic priming with Rif and PB, followed by additional MCT toxicity (Figure 1A). The resultant hepatotoxicity led to lethargy, suggesting encephalopathy, and mortalities occurred over the first week in 14 of 20 (70%) animals versus none in the 20 sham-treated control animals (0%; P = 0.0007, log-rank test) (Figure 1B). The serum alanine aminotransferase and total serum bilirubin were higher after 1 to 3 days. These abnormalities persisted, albeit at lower levels, for 10 to 14 days (Figure 1, C and D). Hepatic necrosis was present at both early and later times (Figure 1E). Immunostaining for CD31 revealed vascular injury and depletion of LSECs (Figure 1E). This effect of MCT in causing LSEC damage corroborated previous findings using electron microscopy in rats.²⁸ This effect of MCT in liver damage was significant because rats given Rif/PB alone without MCT exhibited neither morbidity nor mortality (data not shown). Thus, pro-oxidant Rif/PB priming redirected MCT toxicity in hepatocytes besides LSECs, like that in mice,²⁴ to produce ALF.

Liver injury in rifampicin (Rif)/phenobarbitone (PB)/monocrotaline (MCT)–treated rats. A: Experimental timeline for treatments, including Rif/PB priming over 3 days (d) followed by MCT on d3 and analysis of outcomes, including blood and liver histology assays in experimental groups and controls. Samples were pooled for reporting of early (d1 to d3) and late (d10 to d14) intervals. B: Survival curves showing mortalities during first several days in Rif/PB/MCT group. C and D: Serum alanine aminotransferase (ALT) and total serum bilirubin over 14 days versus controls. E: Representative microphotographs for hepatic parenchymal necrosis [hematoxylin and eosin (H&E) staining; **top panels**] or loss of liver sinusoidal endothelial cells (CD31 staining; brown color, plus toluidine blue nuclear counterstain; **bottom panels**). **Black lines** separate healthy and necrotic liver areas, including those showing central veins (CVs) and perivenous injury. Charts on right supply morphometric data for ≥60% parenchymal necrosis after 1 or 10 to 14 days; and decreases in CD31⁺ areas by ≥80% versus controls. n = 20 each for experimental groups and controls (A). ∗P < 0.05 (analysis of variance and Bartlett post hoc test). Scale bars = 40 μm (E). Original magnifications, ×40 (E, **top panels**); ×400 (E, **bottom panels**). ALF, acute liver failure.

Profiling of Stress and Toxicity Pathway Genes Points to Hepatic DNA Damage and Inflammation

In Rif/PB/MCT-treated animals, 64 of 84 (76%) and 20 of 84 (24%) genes were ≥1.5-fold up or down versus healthy controls after 1 to 3 and 10 to 14 days, respectively (n = 3 each; P < 0.05, χ² test) (Supplemental Table S1).

From the canonical pathways annotated in IPA for mRNAs using –log (P values) of 8.5 and 5, respectively, and false discovery rate <5% at 1 to 3 or 10 to 14 days after Rif/PB/MCT, the top ontologies were as follows (Supplemental Figure S1A). After 1 to 3 days, dysregulation of DDR involved Atm Signaling and Role of Brca1, along with Atm-related downstream processes involving role of Chk proteins in cell cycle checkpoint control, senescence pathway, and p53 signaling. The inflammatory processes involved multiple cell types including: role of protein kinase R in interferon induction and antiviral response; high mobility group box 1 signaling associated with TNF activation; altered T- and B-cell signaling in rheumatoid arthritis; and others, as indicated. After 10 to 14 days, injury and inflammation gained more prominence, including hepatic fibrosis/hepatic stellate cell activation; differential regulation of cytokine production in macrophages and T-helper cells by IL-17A and IL-17F; and cross talk between dendritic cells and natural killer cells. This was consistent with the top toxicology lists at either early or late times after Rif/PB/MCT for the categories of renal, liver, or cardiac necrosis/cell death, increases in liver damage, and decreases in transmembrane potential of mitochondria (Supplemental Figure S1B).

DDR was characterized through an unsupervised approach in IPA for genes in Atm Signaling (n = 10), Chk proteins in cell cycle checkpoint control (n = 8), and senescence pathway (n = 12) (Supplemental Figure S2A). After 1 to 3 days of Rif/PB/MCT, prominent were DNA double-stranded break sensor, Nbn (or nibrin), and repair, Mre11, genes, as well as the mediator of p53-directed G₀/G₁ and G₂/M cell cycle checkpoints, Cdkn1A (or p21). Moreover, the upstream Atm regulator network held 20 nodes, including contributors to inflammatory cytokine signaling and cell survival or proliferation (Supplemental Figure S2B). Similarly, hepatic injury and inflammation genes, including hepatic fibrosis/stellate cell activation (n = 13), or Hmgb1 signaling (n = 9), revealed major chemokines, cytokines, growth factors, or receptors (Supplemental Figure S2C). Among these, Tnf, was highly expressed, and its upstream regulator network included 18 nodes, including ones involved in cell survival or proliferation (Supplemental Figure S2D). The prediction legend is included for the putative relationships in network nodes (Supplemental Figure S2E). These aspects of Rif/PB/MCT hepatotoxicity were expected to correspond to tissue changes.

Tissue analysis identified presence of myeloperoxidase-positive activated PMNs as among early indicators of the innate immune response following Rif/PB/MCT injury (Figure 2A). The PMNs and monocytes are major contributors to cytokines, chemokines, and receptors during liver injury,⁷^,³³ and produce oxidative stress,³ which was reflected in the onset of nitrotyrosine adducts and GGT expression (Figure 2, B and C). Moreover, γH2AX, the Atm-related sensor of DNA double-stranded breaks, was widely expressed, which indicated impairments in DDR (Figure 2D).

Histologic analysis of liver inflammation and injury after 1 day (d) in rifampicin (Rif)/phenobarbitone (PB)/monocrotaline (MCT)–treated rats or healthy controls. A: Myeloperoxidase (MPO)–positive activated neutrophils in periportal area following Rif/PB/MCT injury (dark color; **arrow** in **right panel**). B: Nitrotyrosine (NT) adducts in cytoplasm of injured hepatocytes (dark color; **arrow** in **right panel**). C: GGT in bile ducts from healthy controls (red color; **left panel**) and in hepatocytes after injury (**arrow**; **right panel**). D: Expression of γH2AX in hepatocyte nuclei after injury (**arrow**; **right panel**). Graphs on right provide event quantitation. ∗P < 0.05 (t-tests). Scale bars = 40 μm (A–D). Original magnification, ×400 (A–D). ALF, acute liver failure.

For cell therapy rescue in this ALF model, outcomes after transplanting syngeneic healthy hepatocytes were examined (Figure 3A). Cell therapy produced survival after Rif/PB/MCT in 15 of 20 versus 8 of 20 controls receiving microcarriers alone (P < 0.03, log-rank test) (Figure 3B). In cell recipients at 2 to 3 days after onset of ALF, serum alanine aminotransferase and total serum bilirubin decreased 2.6- and 4.7-fold, respectively (Figure 3C). The prothrombin times in Rif/PB/MCT-treated rats were 5 ± 3-fold and 3 ± 2-fold above normal after 2 to 3 and 10 to 14 days, respectively (P < 0.001). By contrast, in animals subjected to cell transplantation, prothrombin times decreased to 1.6 ± 1-fold and 1.5 ± 1-fold above normal after 2 to 3 and 10 to 14 days, respectively, which differed from sham-treated animals (P < 0.001), but not healthy controls (P > 0.05).

Outcomes in rifampicin (Rif)/phenobarbitone (PB)/monocrotaline (MCT)–treated rats with or without transplantation (Tx) of healthy hepatocytes. A: Experimental outline showing transplantation of 2 × 10⁷ syngeneic hepatocytes in peritoneal cavity 1 day (d) after completing Rif/PB/MCT injury protocol, followed by outcomes analysis, including blood and tissue sampling. B: Survival curves showing mortality decreased in animals receiving cells versus microcarriers alone, 5 of 20 versus 12 of 20, respectively. C: One day after cell transplantation, serum alanine aminotransferase (ALT) and total serum bilirubin levels were lower than in microcarrier-treated controls by 2.6- and 4.7-fold, respectively. D–H: Similarly, 1 day after cell transplantation, hepatic necrosis was 2-fold lower (D), the loss of CD31⁺ sinusoidal endothelial cells decreased by 5-fold (E), the number of myeloperoxidase (MPO)⁺ neutrophils lowered by 3.5-fold (F), hepatic nitrotyrosine adducts decreased by 20-fold (G), and GGT⁺ hepatocytes were 2.4-fold less than in microcarrier-treated controls (H). ∗P < 0.05 (analysis of variance and Bartlett post hoc test). ALF, acute liver failure.

After cell transplantation, hepatic necrosis decreased, endothelial integrity increased, with less depletion of LSECs, fewer myeloperoxidase-positive PMNs, and nitrotyrosine adducts, and hepatic GGT expression declined (Figure 3, D–H).

The transplanted hepatocytes successfully engrafted and were present within microcarrier conglomerates recovered from the peritoneal cavity after 10 to 14 days (Figure 4A). Transplanted cells were adjacent to the microcarriers and surrounded by the host-derived stroma, including capillaries and mesenchymal cell types, as expected. These hepatocytes showed characteristic nuclear and cytoplasmic morphology. Moreover, transplanted hepatocytes showed presence of G6P and glycogen, consistent with their metabolic ability for supporting survival in ALF (Figure 4, B and C). The presence of albumin expression verified protein synthetic ability of transplanted hepatocytes (Figure 4D). Also, staining for Ki-67 as a marker of proliferation in cells with or without albumin (Figure 4, E and F) identified proliferation in native stromal cells and not in transplanted hepatocytes after 10 to 14 days, which suggested their paracrine effects were responsible for regeneration in the native liver.

Engraftment and function of transplanted hepatocytes in peritoneal cavity. Histology of transplanted cell–microcarrier conglomerates in animals after 10 to 14 days. A: Hematoxylin staining showing transplanted hepatocytes adhering to or next to microcarriers (mc) within stroma and capillaries originating from the host. B and C: Histochemical staining, showing presence of hepatic functions in transplanted hepatocytes with G6P (dark color; B), and glycogen content (purple stain; C). D and E: Immunostaining showing transplanted hepatocytes expressing albumin (cytoplasmic red staining; D), whereas colocalization of albumin and Ki-67 (E) showed proliferation in stromal cells (green Ki-67⁺ nuclei; **arrows**), and not in albumin-expressing hepatocytes with red cytoplasm (**arrowheads**). A–E: Representative areas for microcarriers are marked (mc). **Insets:** Electronically magnified views of **boxed areas**. F: Absence of staining in control section incubated without primary antibodies. Scale bars = 20 μm (A–F).

To find evidence for this paracrine support from transplanted hepatocytes, mRNA expression profiles were examined in animals 10 to 14 days after Rif/PB/MCT with cell transplantation or sham treatment versus healthy controls (Supplemental Table S1). Focusing on IPA categories of Liver Necrosis/Cell Death (n = 12), Atm Signaling (n = 5 to 7), or Senescence Pathway (n = 5 and 6), we found cell transplantation lowered or normalized gene expression levels versus Rif/PB/MCT-treated animals (P < 0.05, t-tests) (Supplemental Figure 3A). For liver necrosis and cell death category, up-regulated genes after Rif/PB/MCT included inflammatory cytokines (ie, TNF, Il6, and Serpine1) and apoptosis or necroptosis genes (ie, Fas, Ripk3, and Xbp1). Similarly, Atm signaling or senescence pathway included DNA damage sensing and repair genes (ie, Ddit3, Gadd45a, Gadd45g, Nbn, and Rad51), Atm transducer Chek2, and cell cycle checkpoint regulator Cdkn1a (or p21). Cell transplantation also affected Akt signaling, which is involved in intermediary metabolism, as well as Stat3 signaling, which contributes to hepatic regeneration during toxic injury.²^,¹⁷ After Rif/PB/MCT, Akt and Stat3 signaling were up-regulated, which were normalized by cell transplantation (Supplemental Figure 3B). The upstream regulator networks for Akt and Stat3 in IPA showed disruptions after Rif/PB/MCT injury, including inhibition of Pparg, which directs mitochondrial homeostasis and biogenesis for cell survival and proliferation (Supplemental Figure S3, C–F). Also, Stat3 network suggested effects of Rif/PB/MCT injury for Smad signaling, which directs hepatic differentiation or growth inhibition through transforming growth factor-β and other cytokines. More importantly, cell transplantation restored Akt or Stat3 networks.

To identify signaling pathway activations after ligand-receptor interactions, blood levels of 12 well-expressed cytokines were characterized in healthy animals (Figure 5A). After Rif/PB/MCT at either 1 to 3 or 10 to 14 days, blood cytokine levels differed from healthy animals, including increased (GM-CSF, IFN-γ, Il-1b, Il-6, Il-10, Il-13, and TNF-α) or decreased (G-CSF, Il-1a, and Il-4) (P < 0.05, t-tests) (Figure 5B). After 10 to 14 days of cell transplantation, G-CSF, GM-CSF, IFN-γ, Il-6, Il-10, and Il-12 levels were higher than in sham-treated controls receiving Rif/PB/MCT and in healthy animals (P < 0.05, t-tests) (Figure 5B). These differences in cytokine profiles were significant for Akt-regulated intermediary glucose metabolism, because histochemical staining revealed less G-6-P and glycogen after Rif/PB/MCT, whereas cell transplantation restored these (Figure 5C). Immunostaining showed native hepatocytes in recipients of transplanted cells widely expressed pStat3, which corroborated cytokine receptor–mediated Jak/Stat signaling from G-CSF or GM-CSF, IFN-γ, or ILs. pStat3 expression was absent in Rif/PB/MCT-treated or healthy animals (Figure 5D). Importantly, NPCs within sinusoids also expressed pStat3. These cells included morphologically distinct LSECs, as well as monocytes or Kupffer cells (KCs). The contribution of pStat3 in restoring Atm-mediated DDR was indicated by widespread phosphorylated Atm expression in hepatocytes after Rif/PB/MCT injury, but not after cell transplantation. This aspect of pStat3 and phosphorylated Atm signaling interaction accompanied greater γH2AX expression during hepatic injury and its absence after cell transplantation and liver regeneration. The Ki-67 expression substantiated proliferative activity of hepatocytes in recipients of transplanted cells (Figure 5E). Taken together, the transplanted cells not only rebalanced inflammatory and metabolic consequences, but also elicited multi-level interactions through paracrine factors, including receptor-mediated responses in native hepatocytes and NPCs.

Cell transplantation (Tx)–related effects for plasma cytokines and signaling events in rifampicin (Rif)/phenobarbitone (PB)/monocrotaline (MCT)–treated rats. A: The mean serum levels for healthy rats in pg/mL of 12 cytokines, including interferon (IFN)-γ, multiple ILs, tumor necrosis factor (TNF)-α, and the hematopoietins, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). B: Serum cytokine levels in Rif/PB/MCT-treated rats 1 to 3 or 10 to 14 days (d) after acute liver failure (ALF) and 10 to 14 days following cell transplantation versus healthy rats (P value obtained using t-tests). C: Histochemical staining of liver tissue showing Rif/PB/MCT injury decreased G6P and glycogen (Gly) content and its restoration by cell transplantation. Periportal areas contain more G-6-P (dark color in cytoplasm), whereas glycogen is more abundant in perivenous areas (CVs; purple color in cytoplasm). D: Immunostaining for phosphorylated Stat3 (pStat3; **top panels**), phosphorylated ataxia telangiectasia mutated (pAtm; **middle panels**), and γH2AX (**bottom panels**), showing cell transplantation affected these parameters. In healthy liver, pStat3 was rarely expressed (**arrows**; **left panel**), pAtm was absent, and γH2AX was occasionally found (**arrows**; **left panel**). After hepatic injury, pStat3 was not expressed, whereas pAtm and γH2AX were widely expressed (**arrows**; **middle panels**). Cell transplantation resulted in widespread expression of pStat3 in hepatocytes (**arrows**), as well as sinusoidal endothelial cells (**long arrows**), and monocytes in sinusoids, including Kupffer cells (**arrowheads**). E: Ki-67 immunostaining revealing only occasional liver cells undergoing proliferation in healthy or Rif/PB/MCT-treated animals (**left** and **middle panels**), but cell transplantation led to more Ki-67⁺ cells, including hepatocytes (**arrows**) and sinusoidal cells (**arrowheads**). Graphs on right indicate morphometric quantitation. n = 3 healthy rats (A); n = 3 animals each (C–E). ∗P < 0.05 (analysis of variance and Bartlett post hoc test). Scale bars = 40 μm (C–E). Original magnifications, ×40 (C); ×400 (D and E). PA, portal area.

Discussion

The current ALF modeling in F344 rats verified prior findings in NOD/SCID mice,²³ including liver necrosis, inflammation, and other insults. The role of conjoint drug-induced liver injury and injury from cofactors is significant for human ALF. For instance, hepatotoxicity from APAP, the leading cause of ALF in the Western world,¹ is exacerbated by alcohol,³⁴ nonalcoholic fatty liver disease,³⁵ or cystic fibrosis.³⁶ The evidences of ATM insufficiency, oxidative stress, DNA damage, and lack of liver regeneration support these aspects of human ALF.³ Significantly, during drug toxicity, ATM protein can be depleted through its oxidative inactivation,³ which exacerbates oxidative stress, mitochondrial dysfunction, DNA damage, cell growth arrest, or cell death, whereas inhibition of ATM kinase activity too can amplify drug toxicity.²¹

The intact immunologic system in healthy F344 rats is an advantage for ALF modeling, although innate immune system already contributes to tissue injury, including through granulocytes, KCs, and other NPCs.⁷ Although MCT rapidly depletes LSECs,²⁸ and KCs or hepatic stellate cells can be depleted by gadolinium chloride and clodronate,³⁷ or gliotoxin,³⁸ respectively, removing these cell types can greatly increase susceptibilities for sepsis or other complications, and simultaneously confound liver regeneration. Therefore, this study avoided such manipulations.

Rif/Phen/MCT model in NOD/SCID mice and Rif/PB/MCT model in F344 rats represent ALF following drug-induced liver injury, toxins, ischemia, Cu toxicosis in Wilson disease, or other forms of liver necrosis, rather than those related to immune dysregulations, such as autoimmune hepatitis, viral hepatitis, checkpoint inhibitor toxicities, indeterminate cases, or other immune insults.¹ Besides, fundamental disparities in immune systems in rodents and humans make it unlikely ALF arising from cytotoxicity from lymphocytes, natural killer cells, T cells, or other cells, could be simply modeled in NOD/SCID mice or F344 rats. Nonetheless, hepatic necrosis, inflammation, and growth arrest in these instances should certainly bear on morbidity, and Atm pathway-related processes will remain important for the pathophysiology and reversal of ALF.

The benefits of cell transplantation emanated from the composite of metabolic support and replacement of regenerative factors. The blood levels of candidate inflammatory cytokines did not always decline, because some ILs, including IL-6, and TNF-α, remained elevated. However, levels of other ILs, IFN-γ, G-CSF, and GM-CSF increased in support of the idea that rebalancing of multiple cytokines may be even more significant than correcting individual ones. The biological effects of cytokines clearly included Atm, Stat3, and Akt signaling in hepatocytes and NPCs, along with their consequences for hepatic metabolism, DDR, and proliferation. Thus, these effects in liver cells provide unique information for cell activations in ALF beyond those observed at transcriptomic levels in either whole tissue or single cells.²^,³^,⁵^,⁶ This is important because mRNA and protein levels are often incongruent.¹² Nonetheless, the mRNA expression arrays did reveal critical DDR-related events and processes, whereas regulator networks and downstream effector events in tissues added value to this molecular information.

The ability of healthy hepatocytes themselves to secrete multiple cytokines, growth factors, receptors, and other molecules, including G-CSF, GM-CSF, IFN-γ, IL-10, or IL-12,² should be significant for pathobiological mechanisms. For example, IFN-γ contributes to innate immune responses for microbial clearance, and organ or cell allografts.⁷ Other cytokines inform on the context of immune response in cell types, including hepatocytes or NPCs (eg, TNF-α, CCR5, and CXCR3 chemokine receptors, as well as IL-12, may amplify IFN-γ and IFN-γ–stimulated genes to recruit T and regulatory T subsets for either protective or injury effects.39, 40, 41 Similarly, IL-10 and the related IL-20 cytokine subfamily can exert liver protection or injury effects.⁴² Similarly, the effects of GM-CSF in maturing granulocytes (PMNs and eosinophils) or monocyte/macrophage lineages should naturally extend to liver-resident KCs or dendritic cells, and infiltrating inflammatory cell types.

Significantly, IFNs, ILs, G-CSF, and GM-CSF share receptor-mediated signaling through JAK/STAT pathways, and STAT3 activation is cytoprotective for hepatocytes.²^,¹⁷ Therefore, in F344 rats with Rif/PB/MCT-induced ALF, activation of Stat3 signaling after hepatocyte transplantation in native hepatocytes, as well as NPCs (including LSECs and KCs), says these cytokines were not only biologically active, but benefited regeneration in the native liver through complex effects. As hepatocyte transplantation will have guided secondary release of cytokines, chemokines, and receptors from native liver cells after injury,⁵ this ought to have further contributed for dampening systemic inflammation. Such potential gains in F344 rats are evidenced by Akt signaling and associated regulators for improvements in intermediary metabolism, and Stat3 pathway intersections for restoration of Atm signaling and DDR. The latter aspect recapitulates cytokine receptor-mediated intersections in STAT3 and ATM pathways for APAP toxicity in HuH-7 human hepatocytes.¹⁷ Multiple other cytokines capable of activating JAK/STAT3 signaling in hepatocytes and adjacent NPCs will likely be relevant for hepatic homeostasis in ALF.

Despite careful evaluations of inflammatory and other cytokines in blood of people with ALF, the significance of such profiles has been elusive for pathophysiology of either liver cell interactions or prognostic algorithms.¹¹ One difficulty involves altered phenotypes, implying activation or inactivation states in multiple liver cell types (eg, as revealed in single-cell transcriptomes of hepatocytes, LSECs, KCs, or hepatic stellate cells after APAP or thioacetamide toxicities).⁵ The NPCs are major contributors to inflammation-related cytokines, but in cases, these can exert anti-inflammatory benefits, as noted for mitigation of hepatic ischemia-reperfusion damage after transplantation of healthy KCs.³⁷ Thus, recapitulating Atm-related injury and recovery in this Rif/PB/MCT rat model of ALF should facilitate liver regeneration studies, as outlined in a working model (Figure 6).

Working model for liver regeneration in acute liver failure. The illustration depicts that severe hepatic injury causes cell-intrinsic damage, along with release of cytokines and other signals from hepatocytes and nonparenchymal cells exerting autocrine and paracrine effects in cell types. A: The consequences of cytokine imbalances will include deleterious engagements of receptors and molecular pathways [eg, to inactivate ataxia telangiectasia mutated (ATM) or STAT3 signaling, leading to worse cell damage and losses culminating in liver failure]. B: Healthy transplanted (Tx) cells providing metabolic support and factors to restore ATM and STAT3 pathways will allow cell damage repair, cell growth, and liver regeneration.

From an applied perspective, restoring hepatic homeostasis in ALF could take the form of multicytokine therapies (eg, benefits from various anti-inflammatory and hepatotrophic factors may be established in Rif/PB/MCT rat model). Correlating potential benefits for cell phenotypes within tissues should be especially informative for acknowledging the effects of administered factors in conjunction with intracellular signaling and liver regeneration events for ALF. The benefits for DDR and cell growth via Atm pathway and other mechanisms should be equally significant. Whether hepatocytes derived from pluripotent or other stem cells, despite their incomplete differentiation states,⁴³ might in conjunction with cytokines be effective for rescue in ALF is another possibility worthy of testing.

As progress is being made in obtaining human xenograft-capable rats (eg, the immunodeficient SRG rat),⁴⁴ establishing the potential for treating ALF in modified Rif/PB/MCT rat model of donor human cells or of xenogenic donor cells, such as those from genetically engineered pigs,⁴⁵ should offer many other opportunities for liver regeneration.

Footnotes

Supported by the National Institute of Diabetes and Digestive and Kidney DiseasesR01-DK071111 (S.G.), R01-DK088561 (S.G.), P30-DK41296 (Liver Center), and P30 DK020541 (Diabetes Center).

Disclosures: None declared.

Current address of Y.S., Homology Medicines, Bedfordshire, MA.

Supplemental material for this article can be found at http://doi.org/10.1016/j.ajpath.2022.10.003.

Supplemental Data

Supplemental Figure S1 — Hepatic expression of stress and toxicity genes according to annotations in Ingenuity Pathway Analysis (IPA) at early [1 to 3 days (d)] or late (10 to 14 days) times after acute liver failure (ALF) versus vehicle-treated controls. A: Top 15 canonical pathways represented in comparisons for early and late times at –log (P values) of 8.5 and 5, respectively, and false discovery rate of <5% by Fisher exact test are indicated. Noteworthy were DNA damage response pathways at early time, including Atm Signaling and Role of Brca1 in DNA Damage Response; Role of Chk Proteins in Cell Cycle Checkpoint Control, p53 Signaling, and Senescence Pathway at –log (P values) of 12.8, 11.9, 11.4, 9.5, and 10.8, respectively. The inflammatory signaling included Role of PKR in Interferon Induction and Antiviral Response; Altered T- and B-Cell Signaling in Rheumatoid Arthritis; and HMGB1 Signaling, which is associated with tumor necrosis factor (TNF), at –log (P values) of 15.3, 9.8, and 9.1, respectively. After 10 to 14 days, additional inflammatory and injury pathways gained prominence, including Hepatic Fibrosis/Hepatic Stellate Cell Activation; Differential Regulation of Cytokine Production in Macrophages and T-Helper Cells by IL-17A and IL-17F; Cross Talk between Dendritic Cells and Natural Killer Cells, and others, at –log (P values) of 11.7, 6.3, and 6.0, respectively. B: Top toxicology gene lists represented in this mRNA expression profiling reproduced at both early and late times, Renal, Liver, or Cardiac Necrosis/Cell Death, Increases in Liver Damage, and Decreases in Transmembrane Potential of Mitochondria. Total n of genes, those in data sets, and P values for categories are indicated. n = 3 each for ALF and vehicle-treated control groups.

Supplemental Figure S2 — Activation of *Atm* or injury and inflammation pathways annotated by Ingenuity Pathway Analysis (IPA) 1 to 3 days after rifampicin (Rif)/phenobarbitone (PB)/monocrotaline (MCT) injury versus vehicle-treated healthy controls. A: Alignment of differentially expressed genes for Ataxia Telangiectasia Mutated (Atm) Signaling, Chk Proteins in Cell Cycle Checkpoint Control, including *Chek2*, which directly mediates *Atm* signals, and Senescence Pathway indicates cumulative gene expression (chart on left) and individual expression-level differences (heat maps on right). The differences are noteworthy for up-regulation of *Nbn* (or *nibrin*), a sensor for DNA double-stranded breaks, and *Mre11*, which repairs DNA double-stranded breaks, as well as *Cdkn1a* (or *p21*), which imparts *p53*-mediated G₀/G₁ and G₂/M checkpoints. B: Predicted *Atm* upstream regulator network at z-score of 1.6, P value for overlap in networks of 1.24 × 10⁻¹⁶, and 20 nodes, suggests potential for interferences in cell survival or cycling via *Stat3* and *NF-kb* genes transducing cytokine signaling. C: Cumulative mRNA expression for Hepatic Fibrosis/Stellate Cell Activation or Hmgb1 Signaling (chart on left), and individual gene expression levels (heat maps on right). Among cytokines and mediators, *Tnf* was highly significant. D: Schematic representation of tumor necrosis factor (TNF) regulator network at z-score of 4.7, P value for overlap of 1.40 × 10⁻²³, and 18 nodes, indicating potential for exerting widespread cell survival and proliferation effects via multiple regulators. E: Prediction legend for gene interactions. n = 3 each for Rif/PB/MCT injury and vehicle-treated healthy controls; n = 10 for Atm Signaling (A); n = 8 for Chk Proteins in Cell Cycle Checkpoint Control (A); n = 12 for Senescence Pathway (A); n = 13 for Hepatic Fibrosis/Stellate Cell Activation (C); n = 9 for Hmgb1 Signaling (C).

Supplemental Figure S3 — Effects of transplanted (Tx) hepatocytes 10 to 14 days (d) after rifampicin (Rif)/phenobarbitone (PB)/monocrotaline (MCT)–induced acute liver failure (ALF) versus healthy controls on liver necrosis, cell death, and cell fate. A: Ingenuity Pathway Analysis (IPA)–derived representations for categories of liver necrosis/cell death, *Atm* signaling, or senescence pathway with n of 12 each, 5 and 7, and 5 and 6, respectively. Genes in *Atm* signaling or senescence pathway overlap because these exert shared mechanisms. The expression levels of individual genes are indicated in boxes for heat maps. The chart provides grouped gene expression levels. After cell transplantation, gene expression levels decreased in each category. The necrosis and cell death category includes inflammatory cytokines (ie, *TNF*, *Il6*, and *Serpine1*), regulators of apoptosis or necroptosis (ie, *Fas*, *Ripk3*, and *Xbp1*); the Atm signaling, or senescence pathways, include DNA damage sensors and repair genes (ie, *Ddit3*, *Gadd45a*, or *Gadd45g*, *Nbn*, and *Rad51*), along with the *Atm* transducer, *Chek2*, and the cell growth regulator, *Cdkn1a* (or *p21*). B: Expression of genes for *Akt (n* = 24 and 32) and *Stat3* signaling (n = 26 and 23), which are major contributors to intermediary metabolism and cytokine signaling, respectively. Cell transplantation normalized up-regulated genes in these signaling pathways after Rif/PB/MCT injury. C: Upstream *Akt* regulator network from IPA with z-score of 2.47, P for overlap of 1.64 × 10⁻²², and 19 regulator nodes, indicating Rif/PB/MCT injury, resulting in down-regulation of *Pparg*, which directs mitochondrial biogenesis and functionality for cell survival and proliferation. D: Upstream *Akt* regulator network after cell transplantation with z-score of 2.46, P for overlap of 3.22 × 10⁻¹⁶, and 25 nodes, indicating restorations in *Pparg* and other regulators. E and F: Upstream *Stat3* regulator network after Rif/PB/MCT injury reiterates the negative regulation for *Pparg*, along with additional *Smad* signaling dysregulations involved in cell differentiation and growth (E), whereas after cell transplantation these regulators are restored (F). Prediction legend for gene interactions follows that in Supplemental Figure S2. n = 3 each for ALF and healthy controls. ∗P < 0.05 (t-tests).

Supplemental Table S1

mmc1.docx^{(30.3KB, docx)}

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Associated Data

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Supplementary Materials

Supplemental Table S1

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PERMALINK

Hepatocyte Transplantation Rebalances Cytokines for Hepatic Regeneration in Rats with Ataxia Telangiectasia Mutated Pathway-Related Acute Liver Failure

Fadi-Luc Jaber

Yogeshwar Sharma

Sanjeev Gupta

Abstract