Cytokine-Induced Liver Hepatotoxicity of Trovafloxacin in Co-Culture ofHepatocytes and Kupffer Cells
12221
Kelly Rose , Kimberly Freeman , Stephen S. Ferguson , Rafal P. Witek , Edward L. LeCluyse

1. The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709, United States 2. Life Technologies, Durham, NC, United States

Immune-mediated chemical-induced
hepatotoxicity, i.e. indirect hepatocellular toxicity
resulting from immune cells activating liver
inflammatory responses, is often overlooked as apotential mode of action due to unavailability ofappropriate in vitro models. Kupffer cells are thelargest population of resident macrophages in theliver and thus play a critical role in immunemediated hepatotoxicity and liver injury. For thisreason, we have established a co-culture system ofrat primary hepatocytes and Kupffer cells that canbe used to model chemical-induced immune
reactions resulting in acute hepatotoxicity. Coculture of hepatocytes and Kupffer cells may
represent a powerful in vitro tool to predict adverse
liver effects resulting from indirect adaptive
immune reactions during chemical exposure.

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Figure 3. Treatment of co-cultures with LPS (1 µg/ml) results in an inhibition of CYP3A activity which isreversed after co-treatment with TVX (50 µM). A. At 24 hr LPS co-culture treatment groups begin toshow a decrease in CYP3A activity (34% less than control) which is reversed when TVX is added alongwith the LPS (14% less than control). This pattern of inhibition of activity with LPS then recovery ofactivity with TVX/LPS continues to a greater degree over 48 hr with 74% inhibition with LPS, 6% inhibitionfor TVX/LPS (B) and 72 hr with 85% decrease in CYP3A activity with LPS recovering to 44% when TVX isadded along with LPS (C) . Inhibition of CYP3A activity by LPS and recovery by adding TVX was notobserved in hepatocyte monocultures to the degree observed in co-cultures.

DConclusion
Co-cultures of hepatocytes and Kupffer cells can beused to predict chemical-induced immune reactions
that result in drug hepatotoxicity. As shown in thiswork, co-cultures showed blunted IL-6 response andincreased TNF-α production that resulted in hepaticnecrosis and concurrent increases in LDH anddecreases in ATP levels indicating cytotoxicity. Thisdata supports use of hepatocytes and Kupffer cells inco-cultures as a powerful in vitro tool to evaluate theeffects of drug toxicity that are not apparent using invitro models of monocultured hepatocytes.

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100CFigure 8: Increased necrotic cell deathin co-cultures vs. hepatocytes following
48 hr TVX/LPS treatment.
A.
Hepatocytes treated with LPS (1µg/mL) only; B. Hepatocytes treatedwith TVX/LPS (200 µM, 1 µg/mL). C.
Co-cultures treated with LPS (1 µg/mL)
only; D. Co-cultures treated withTVX/LPS (200 µM, 1 µg/mL). Cocultures clearly exhibit effects ofincreased
cytokinelevels
anddecreased ATP levels compared tohepatocyte monocultures.

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3000CFigure 7. Treatment of co-cultures for 48 hr with LPS (1 µg/ml) and Trovafloxacin(TVX, 0-200 µM) has a dose dependent effect on ATP levels. Graph of ATP levels inhepatocytes and co-cultures after 48 hr of treatment with LPS (1 µg/ml) and TVX (0200 µM). ATP levels decrease for both cultures at 50 µM TVX and exhibited a muchsharper drop in ATP levels for co-cultures alone, likely due to the changing shifts incytokine profiles with increasing TVX dose. Note that Kupffer cells alone had ATP levels
below levels of detection and are not shown.

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Figure 5: Increased necrotic cell deathfollowing 48 hr TVX/LPS treatment. A.
Co-cultures treated with LPS (1 µg/mL)
only; B. Co-cultures treated withTVX/LPS (50 µM, 1 µg/mL). Noteincreased appearance of necrotic foci(arrows) as compared with LPS-onlygroup. This suggests the potential
involvement of deregulated cytokines inthis model of hepatotoxicity.

*+Figure 2. Treatment of co-cultures with LPS (1 µg/ml) and Trovafloxacin (TVX, 50 µM) deregulates cytokine
production, leading to an abnormal accumulation of TNF-α and the inhibition of IL-6 production. A. At 24
hr, TVX/LPS and LPS co-culture and Kupffer cells treatment groups display similar ratios of IL-6 to TNF-αproduction. This ratio changes dramatically at 48 hr (B) and 72 hr (C) where in LPS only treatment, IL-6
increases and TNF-α remains stable, whereas in TVX/LPS, TNF-α remains abnormally high to IL-6 whoseproduction is inhibited. This suggests a reversion of the TNF-α to IL-6 ratio where hepatotoxic TNF-α
predominates, and CYP3A modulation by IL-6 is absent. Significance: + P<0.1, * P<0.05, ** P<0.01

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Figure 4. Treatment of co-cultures with LPS (1 µg/ml) and Trovafloxacin (TVX, 50 µM) affects LDH andATP levels. Representative graph of LDH and ATP levels in hepatocytes, co-cultures, and Kupffer cells
after 48 hr of treatment with LPS (1 µg/ml) and/or TVX (50 µM). A. LDH levels in hepatocytes andKupffer cells show no change after treatment, while treatment of co-cultures with LPS and TVX/LPS
result in an increase of 29% and 46% respectively in LDH levels over no treatment controls. B. Similarly,
ATP levels were not affected in hepatocytes or Kupffer cells after treatment with LPS and/or TVX, whileco-cultures exhibited a drop in ATP (28% compared to no treatment controls) when treated withTVX/LPS. Note that 24 and 72 hr treatments exhibited comparable ATP and LDH results.

BLuciferin IPA (LU)

Cytometric analysis of primary Kupffer cells
displayed at least 90% purity of two distinctive
subpopulations of CD68/CD163 cells. Twenty four,48, and 72 hr after LPS treatment, cytokine
analysis of hepatocyte and Kupffer cell co-cultures
for IL-6 and TNF-α was performed to analyze theeffect of LPS on hepatic metabolism. Inflammation
markedly down-regulated activity of CYP3A 74%
and 85% at 48 and 72hr after LPS treatment,respectively. Inhibition of metabolism correlatedwith IL-6 up-regulation. This response was blunted
in co-treatment with LPS and TVX showing areversal of inhibition of CYP3A activity andsignificantly lower production of IL-6; however, TNF-α production was unaffected. A subsequent study
utilizing LPS and increasing amounts of TVX
exhibited the same pattern of reversal of inhibition
of CYP3A activity and shifting IL-6/TNF-α ratios in adose-dependent fashion. ATP and LDH levelscorrelated with observed necrotic cytotoxicity inhepatocytes after TVX/LPS treatment.

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12Methods
Rat hepatocytes and Kupffer cells were obtained
from the Hamner Institutes or Life Technologies.
Kupffer cells and hepatic co-cultures were culturedinAdvanced
DMEM
supplementedwith
Penicillin/Streptomycin, GlutaMax, and 15 mM
HEPES. For plating media, an additional 10% FBS
was used. Cells were analyzed for purity using theAttune Acoustic Focusing Cytometer. Kupffer cellmorphological assessments with CD68 and CD163
wereperformedutilizingstandard
IHC
methodologies. Rat hepatocytes, Kupffer cells, andco-cultures were dosed for 24-72 hr with 1 µg/ml
E. coli (0127:B8) LPS and various concentrations ofTVX. CYP3A activities were measured using P450Glo Luciferin IPA assayTM , ATP levels were measured
using Cell-Titer GloTM and LDH levels weremeasured using CytoTox-OneTM assay (Promega).
Cytokines were assessed by ELISA or Luminex®
beads for IL-6 and TNF-α (Life Technologies).

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LDH Activity (LU)

Introduction

Figure 1. Characterization of isolatedand cryopreserved rat Kupffer cells.
A. CD68 (ED1) and B. CD163 (ED2)immuno-stained cells that support
their macrophage lineage. Additional
flow analysis reveals that bothmarkers (CD68 in C and CD163 in D)
are represented by the majority of
the cells, suggesting very high purityof Kupffer cells (>90%).

Trovafloxacin (µM)
Figure 6. Treatment of co-cultures for 48 hr with LPS (1 µg/ml) and Trovafloxacin (TVX, 0-200µM)
deregulates cytokine production, leading to an abnormal accumulation of TNF-α and the inhibition of IL6 production. A. Increasing amounts of TVX in co-cultures result in a 2-4 fold increase in CYP3A activity
over no TVX control with while hepatocyte monocultures exhibit a 1.5 to 2 fold increase of CYP3Aactivity only at the highest doses of TVX. B. IL-6 levels in co-cultures gradually decline with increasing
TVX dose, appearing to cause a reversal of effects of LPS on CYP3A levels in co-cultures. Kupffer cells
display a reduced but similar trend in IL-6. C. TNF-α levels show a slight increase and gradual declinewith both co-cultures and Kupffer cells, suggesting TNF-α levels predominate over IL-6 levels withincreasing TVX dose for co-cultures and Kupffer cells to a lesser degree. Decreasing ATP levels at 50,100, and 200 µM TVX indicate concurrent cytotoxic effects on these measurements.

References
Available upon request.

Acknowledgements
This project was supported by funding from the LongRange Research Initiative (LRI) of the American
Chemistry Council (ACC). Thanks to Dr. Jessica Bonzo
for her editorial assistance and review of the poster.
Thanks to Dr. Jingtao Lu for presenting the poster.