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. 2024 May 30:15:1335836.
doi: 10.3389/fphar.2024.1335836. eCollection 2024.

Assessing immune hepatotoxicity of troglitazone with a versatile liver-immune-microphysiological-system

Quanfeng Deng  1 , Youlong Yang  1 , Yuangui Liu  1 , Mengting Zou  1 , Guiyuan Huang  1 , Shiqi Yang  1 , Lingyu Li  1 , Yueyang Qu  1 , Yong Luo  2 , Xiuli Zhang  1   3
Affiliations

Assessing immune hepatotoxicity of troglitazone with a versatile liver-immune-microphysiological-system

Quanfeng Deng et al. Front Pharmacol. .

Abstract

Drug-induced liver injury is a prevalent adverse event associated with pharmaceutical agents. More significantly, there are certain drugs that present severe hepatotoxicity only during the clinical phase, consequently leading to the termination of drug development during clinical trials or the withdrawal from the market after approval. The establishment of an evaluation model that can sensitively manifest such hepatotoxicity has always been a challenging aspect in drug development. In this study, we build a liver-immune-microphysiological-system (LIMPS) to fully demonstrate the liver injury triggered by troglitazone (TGZ), a drug that was withdrawn from the market due to hepatotoxicity. Leveraging the capabilities of organ-on-chip technology allows for the dynamic modulation of cellular immune milieu, as well as the synergistic effects between drugs, hepatocytes and multiple immune cells. Through the LIMPS, we discovered that 1) TGZ can promote neutrophils to adhered hepatocytes, 2) the presence of TGZ enhances the crosstalk between macrophages and neutrophils, 3) the induction of damage in hepatocytes by TGZ at clinically relevant blood concentrations not observed in other in vitro experiments, 4) no hepatotoxicity was observed in LIMPS when exposed to rosiglitazone and pioglitazone, structurally similar analogs of TGZ, even at the higher multiples of blood drug concentration levels. As an immune-mediated liver toxicity assessment method, LIMPS is simple to operate and can be used to test multiple drug candidates to detect whether they will cause severe liver toxicity in clinical settings as early as possible.

Keywords: hepatotoxicity; innate immune; liver injury; microphysiological systems; organ chip.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Adhesion of dHL60 cells in the LIMPS. (A) Adhesion of dHL-60 cells on the HepG2 cells under different circumstances. (B) Partial schematic diagram of the LIMPS, dynamics of the adhesion of the dHL-60 cells (n = 3, **p < 0.01, ***p < 0.001). (C) Dashed circles indicate red dHL-60 cells adhesion causing the apoptosis of green HepG2 cells.
FIGURE 2
FIGURE 2
The effect of the dHL-60 cells on the hepatotoxicity due to TGZ in the LIMPS. (A) Live/death staining with calcein AM/PI/HOE33342 of all live cell in the LIMPS. (B) The relationship between the all live cell proportion and TGZ/dHL-60 cells. (C) The relationship between the absolute all live cell quantity and TGZ/dHL-60 cells. (D) The relative mRNA expression inside the dHL-60 cells (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)
FIGURE 3
FIGURE 3
The effect of the NK-92 cells on the hepatotoxicity due to TGZ in the LIMPS. (A) Live/dead staining of the HepG2 cells. (B) Quantitative analysis of the fluorescence images in (A). (C) Inhibitory effect of troglitazone on the NK-92 cells. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).
FIGURE 4
FIGURE 4
The effect of the THP-1 cells on the hepatotoxicity due to TGZ in the LIMPS. (A) Variation of the HepG2 cell numbers with THP-1 cell numbers with or without TGZ presence (HepG2: green, THP-1 cells not shown). (B) The enlarged view of the morphology of the HepG2 cells with or without the presence of THP-1 cells (5 × 105). (C) The secretion of the IL-1β, TNF-α, IL-6 and IL-10 with or without TGZ (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).
FIGURE 5
FIGURE 5
The effect of the co-presence of THP-1 and dHL-60 cells on the hepatotoxicity due to TGZ in the LIMPS. (A) Left: the fluorescence of HepG2-GFP with or without TGZ, THP-1 and dHL-60 cells. Right: quantitative analysis of the left images. (B) Secretion of the factors (IL-1β, TNF-α, IL-6, IL-10) under the different circumstances. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)
FIGURE 6
FIGURE 6
Illustrates the comparative toxicities of curcumin, pioglitazone, and rosiglitazone on the LIMPS. (A) Fluorescence image of cell stock in liver channels. (B) Quantitative analysis of the fluorescence images (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).
FIGURE 7
FIGURE 7
Polarization of the THP-1 cells in the LIMPS. (A) Evolvement of THP-1 cells under the stimulation of TGZ in the LIMPS. (B) The morphology of THP-1 cells under different circumstances in the side channel of the LIMPS. (C) CD86 and CD163 immunofluorescence of THP-1 cells under different circumstances. (D) Correlation between the THP-1 polarization and TGZ, HepG2 and dHL-60 cells (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).
FIGURE 8
FIGURE 8
Design and Structure of LIMPS. (A) The illustration of the immune-mediated drug-induced liver injury (left), the design of the LIMPS (middle), and the gravity-based pumping (right). (B) The real LIMPS and the enlarged view of the micro-fences (left), the channel design (middle), and the microfluidic field inside the LIMPS (right).
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