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Research ArticleArticle

Establishment of a Hepatocyte-Kupffer Cell Coculture Model for Assessment of Proinflammatory Cytokine Effects on Metabolizing Enzymes and Drug Transporters

Theresa V. Nguyen, Okechukwu Ukairo, Salman R. Khetani, Michael McVay, Chitra Kanchagar, Wolfgang Seghezzi, Gulesi Ayanoglu, Onyi Irrechukwu and Raymond Evers
Drug Metabolism and Disposition May 2015, 43 (5) 774-785; DOI: https://doi.org/10.1124/dmd.114.061317
Theresa V. Nguyen
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Okechukwu Ukairo
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Salman R. Khetani
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Michael McVay
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Chitra Kanchagar
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Wolfgang Seghezzi
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Gulesi Ayanoglu
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Onyi Irrechukwu
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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Raymond Evers
Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Rahway, New Jersey (T.V.N., R.E.); Hepregen Corporation, Medford, Massachusetts (O.U., O.I.); Colorado State University, Fort Collins, Colorado (S.R.K.); Agios Pharmaceuticals, Cambridge, Massachusetts (M.M.); University Of Massachusetts Medical School, Department of Molecular Medicine, Worcester, Massachusetts (C.K); and Merck Research Laboratories, Department of Bioanalytics, Palo Alto, California (W.S., G.A)
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  • Fig. 1.
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    Fig. 1.

    Kupffer cell CD163 cell surface marker mRNA expression in Hep:KC cultures. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). RT-PCR analysis was performed on samples from days 3, 7, 11, and 15 post-KC addition for mRNA expression of CD163. Results were relative to cultures without KCs (Hep:KC 1:0) (n = 3 ± S.D.). Mean values were significantly different from Hep:KC *1:0 and *#1:0.1 cultures (P < 0.05).

  • Fig. 2.
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    Fig. 2.

    Cytokine secretion in Hep:KC cultures after LPS treatment. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± Kupffer cells (at Hep:KC of 1:0, 1:0.1, and 1:0.4). Secreted proinflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, and IFN-γ) were measured in cell culture media 24 hours after exposure to LPS (50 ng/ml) (n = 3 ± S.D.). Mean values were significantly different from *LPS-untreated Hep:KC cultures of matched cell ratios and *#LPS-treated 1:0 cultures (P < 0.05). aLevels were undetectable.

  • Fig. 3.
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    Fig. 3.

    Cytokine secretion in Hep:KC cultures after IL-6 and IL-1β treatment. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). Secreted proinflammatory cytokines such as IL-8, TNF-α, and IFN-γ were measured in cell culture media 4 days after exposure to IL-6 (A–C) and IL-1β (D–F) (n = 3 ± S.D.). aLevels were undetectable.

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    Fig. 4.

    IC50 of CYP3A4 suppression in Hep:KC cultures after IL-1β and IL-6 incubation. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0 and 1:0.4). After IL-1β and IL-6 (6.25–5000 pg/ml) incubation, CYP3A4 activity levels were measured using the CYP3A4-Glo assay at 4 days after cytokine exposure (A and C) and 6 days after recovery in cytokine-free media (B and D). IC50 values were measured using GraphPad Prism (n = 3 ± S.D.).

  • Fig. 5.
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    Fig. 5.

    Expression of cytokine receptors in Hep:KC cultures. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). RT-PCR analysis was performed on samples from days 3, 7, 11, and 15 for gene expression of (A) IL-6R, (B) IL-1R1, (C) IL-2RB, and (D) IL-23R. Results were relative to cultures without KC (Hep:KC 1:0) (n = 3 ± S.D.). Mean values were significantly different from Hep:KC *1:0 and *#1:0.1 (P < 0.05). aLevels were undetectable (Ct values for 18s housekeeping gene were consistent across all cell samples, but Ct values for IL-2RB were >35 and undetectable for IL-23R).

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    Fig. 6.

    Effects of IL-1 receptor antagonist (IL-1RA) cytokine and anti-IL-1β mAb (αIL-1β) on IL-1β–mediated CYP3A4 suppression. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0 and 1:0.4) and codosed with IL-1β (2.5 ng/ml or 0.1 ng/ml for Hep:KC of 1:0 or 1:0.4, respectively) and either IL-1RA (100–1000 ng/ml) or αIL-1β (10–100 ng/ml). CYP3A4 activity levels were measured using CYP3A4-Glo assay at 4 days after treatment (n = 3 ± S.D.). *Mean values were significantly different from IL-1β-untreated matched Hep:KC cultures (P < 0.05).

  • Fig. 7.
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    Fig. 7.

    Cytokine secretion in Hep:KC cultures resulting from IL-1β, IL-2, and IL-23 exposure. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). Secreted proinflammatory cytokines such as IL-8 (A), TNF-α (B), and IFN-γ (C) were measured in cell culture media 4 days after cytokine treatment (n = 3 ± S.D.). aLevels were undetectable.

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    Fig. 8.

    CYP3A4 activity in Hep:KC cultures after IL-2 and IL-23 treatment. Hepatocytes were cultured alone (in micropatterned cocultures with 3T3-J2 fibroblasts) ± KCs (at Hep:KC of 1:0, 1:0.1, and 1:0.4). CYP3A4 activity levels were measured after 4 days of IL-2 (A) and IL-23 (B) treatment (n = 3 ± S.D.). *Mean values were significantly different from IL-2-untreated Hep:KC cultures of matched cell ratios (P < 0.05).

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    Fig. 9.

    Gene expression of CYP3A4 and CRP in Hep:KC cultures after cytokine treatments. Hepatocytes were cultured (in micropatterned cocultures with 3T3-J2 fibroblasts) ± Kupffer cells (at Hep:KC of 1:0 and 1:0.4). After IL-1β (A and B) and IL-6 (C and D) incubation (6.25–5000 pg/ml), CYP3A4 and CRP mRNA levels were assessed by RT-PCR (n = 3 ± S.D.).

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    TABLE 1

    Effects of IL-1β and IL-6 on metabolism enzymes mRNA expression in Hep:KC (1:0 and 1:0.4) cell cultures

    Metabolism EnzymesExposure to IL-1β (6.25–5000 pg/ml), 4 DaysExposure to IL-6 (6.25–5000 pg/ml), 4 Days
    EffectsaKC-Dependent
Effects?EffectsaKC-Dependent
Effects?
    CYP1A1↔—↔—
    CYP1A2↓Yes↓—
    CYP2A6ND—ND—
    CYP2B6↔—↓—
    CYP2C8↓Yes↓—
    CYP2C9↓Yes↓—
    CYP2C9↔—↔—
    CYP2C19ND—↔—
    CYP2D6↔—↔—
    CYP2E1↔—↔—
    CYP3A4↓Yes↓—
    GSTA1↓bYes↓—
    GSTA2↓bYes↓—
    SULT1A1ND—ND—
    SULT1A2ND—ND—
    UGT1A1↓Yes↓—
    UGT2B7↓bYes↓—
    • ↵a Effects were noted as noneffect (↔) or downregulated (↓) ≤50% of untreated control in a concentration-dependent manner. Samples with Ct value >32 were designated as nondetectable (ND).

    • ↵b Effects were observed only in Hep:KC 1:0.4 but not 1:0 cultures.

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    TABLE 2

    Effects of IL-1β and IL-6 on uptake and efflux transporters mRNA expression in Hep:KC (1:0 and 1:0.4) cell cultures

    TransportersExposure to IL-1β (6.25–5000 pg/ml), 4 DaysExposure to IL-6 (6.25–5000 pg/ml), 4 Days
    EffectsaKC-Dependent
Effects?EffectsaKC-Dependent
Effects?
    NTCP↓bYes↓—
    OAT2↔—↔—
    OATP1B1↓bYes↔—
    OATP1B3↓bYes↓—
    OATP2B1↓—↔—
    OCT1↓—↓—
    BCRPND—ND—
    BSEP↓Yes↓—
    MDR1↔—↔—
    MRP2↔—↔—
    MRP3↔—↔—
    MRP4ND—ND—
    • ↵a Effects were noted as noneffect (↔) or downregulated (↓) ≤50% of untreated control in a concentration-dependent manner. Samples with Ct value >32 were designated as nondetectable (ND).

    • ↵b Effects were observed only in Hep:KC 1:0.4 but not 1:0 cultures.

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    TABLE 3

    Effects of IL-1β and IL-6 on acute-phase proteins mRNA expression in Hep:KC (1:0 and 1:0.4) cell cultures

    ProteinsExposure to IL-1β (6.25–5000 pg/ml), 4 daysExposure to IL-6 (6.25–5000 pg/ml), 4 days
    Effectsa (Highest Observed % Untreated Control)KC-Dependent Effects? Effects (Highest Observed % Untreated Control)KC-Dependent
Effects?
    AAG↑Yes↑—
    1250 ± 94 (1:0)1986 ± 170 (1:0)
    1459 ± 412 (1:0.4)1024 ± 98 (1:0.4)
    CRP↑Yes↑—
    1133 ± 260 (1:0)95,220 ± 12,671 (1:0)
    53,132 ± 15,568 (1:0.4)80,426 ± 25,395 (1:0.4)
    SAA2↑—↑—
    5057 ± 1181 (1:0)18,208 ± 6529 (1:0)
    5319 ± 1688 (1:0.4)10,392 ± 1643 (1:0.4)
    • ↵a Effects were noted as upregulation (↑) of ≥150% of untreated control in a concentration-dependent manner.

Additional Files

  • Figures
  • Tables
  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Data -

      Supplemental Table 1 - Information of Donors Used in Pre-characterization Studies

      Supplemental Figure 1 - Metabolic competency of Hep:KC cultures

      Supplemental Figure 2 - Effects of IL-6 on Hep:KC cultures

      Supplemental Figure 3 - Effects of IL-1-beta on Hep:KC cultures

      Supplemental Figure 4 - Effects of IL-2 on Hep:KC cultures

      Supplemental Figure 5 - Effects of IL-23 on Hep:KC cultures

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Drug Metabolism and Disposition: 43 (5)
Drug Metabolism and Disposition
Vol. 43, Issue 5
1 May 2015
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Research ArticleArticle

Hepatocyte-Kupffer Cell Coculture for Studying Cytokine Effects

Theresa V. Nguyen, Okechukwu Ukairo, Salman R. Khetani, Michael McVay, Chitra Kanchagar, Wolfgang Seghezzi, Gulesi Ayanoglu, Onyi Irrechukwu and Raymond Evers
Drug Metabolism and Disposition May 1, 2015, 43 (5) 774-785; DOI: https://doi.org/10.1124/dmd.114.061317

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Research ArticleArticle

Hepatocyte-Kupffer Cell Coculture for Studying Cytokine Effects

Theresa V. Nguyen, Okechukwu Ukairo, Salman R. Khetani, Michael McVay, Chitra Kanchagar, Wolfgang Seghezzi, Gulesi Ayanoglu, Onyi Irrechukwu and Raymond Evers
Drug Metabolism and Disposition May 1, 2015, 43 (5) 774-785; DOI: https://doi.org/10.1124/dmd.114.061317
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