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Human In-vitro Inflammatory Liver Model Recapitulates Immune-Associated Drug Efects with High Predictivity

ABSTRACT: Immune-mediated drug-induced liver injury is often undermined or not well-studied due to lack of appropriate in vitro models comprised of immune cells and suitable endpoints. Current advances in co-cultures deploying primary human Kupffer cells (PHKCs) or alternative cells such as THP-1 macrophages are limited by their inability of recapitulating drug-induced cytokine responses which are important immune-mediated drug effects. Since human iPSC-derived Kupffer cells (iKCs, PMID: 30497023) functionally resemble PHKCs, we investigated the use of iKCs to mimic drug-specific cytokine responses in vitro. iKCs were co-cultured with hiPSC-derived hepatocytes, which were then subjected to treatment of drugs with known immune-mediated effects. Out of the cytokine measurements including interleukin-6 and tumor necrosis factor α obtained from 19 compounds, 17 showed good in vitro-in vivo correlation (89.5%). Furthermore, co-cultures using THP-1 macrophages instead of iKCs could not recapitulate IL-6 responses upon treatment with paradigm compounds. RNA-Seq was used to explore the interaction between cytokines and drug-induced hepatotoxicity in greater detail for one pair of drugs - trovafloxacin and its non-hepatotoxic analogue, levofloxacin. Our study demonstrates for the first time, the use of iKCs in a physiologically relevant model that captures cytokine responses upon treatment with a wide range of immune-mediated drugs and hence can be very useful in warranting further investigation of new drugs that elicit drug-induced cytokine imbalance.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189320 | GEO | 2024/12/31

REPOSITORIES: GEO

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Project description:Drug-induced liver injury (DILI) is an important clinical problem. Here we used a genomics approach to establish the critical drug-induced toxicity pathways that act in synergy with the pro-inflammatory cytokine tumor necrosis factor (TNF) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and Nrf2 antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. The ER stress was primarily related to PERK and ATF4 activation and subsequent expression of CHOP, which was all independent of TNFÎ± signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision cut human liver slices. Targeted RNA interference studies revealed that while ER stress signaling through IRE1Î± and ATF6 acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNF-induced apoptosis. While inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNF cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNF-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing towards caspase-8-dependent TNF induced apoptosis. We sought to determine the effect of co-exposure of certain drugs associated with idiosyncratic DILI with the cytokine TNFalpha to mimic drug exposure with inflammation signaling in HepG2 cells Human hepatoma HepG2 cells were obtained from American Type Culture Collection (ATCC, Wesel, Germany), cultured in DMEM supplemented with 10% (v/v) FBS, 25 U/mL penicillin and 25 Âµg/mL streptomycin and used for experiments between passage 5 and 20.

Project description:Understanding the mechanisms of host macrophage responses to M. tuberculosis (M.tb.) is essential for uncovering potential avenues of intervention to boost host resistance to infection. Macrophage transcriptome profiling revealed M.tb. infection strongly induced expression of several enzymes controlling tryptophan (Trp) catabolism. This included indole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2), which catalyze the rate-limiting step in the kynurenine pathway, producing ligands for the aryl hydrocarbon receptor (AHR). The AHR and heterodimeric partners AHR nuclear translocator (ARNT) and RELB are robustly expressed, and AHR and RELB levels further increased during infection. Infection enhanced AHR/ARNT and AHR/RELB DNA binding, and stimulated expression of AHR target genes, including that encoding the inflammatory cytokine IL1beta. AHR target gene expression was further enhanced by exogenous kynurenine, and exogenous Trp, kynurenine or synthetic agonist indirubin reduced mycobacterial viability. Comparative expression profiling revealed that AHR ablation diminished expression of numerous genes implicated in innate immune responses, including several cytokines. Notably, AHR depletion reduced expression of IL23A and IL12B transcripts, which encode subunits of interleukin 23 (IL23), a macrophage cytokine that stimulates production of IL22 by innate lymphoid cells. The AHR directly induced IL23A transcription in human and mouse macrophages through near-upstream enhancer regions. Taken together, these findings show that AHR signaling is strongly engaged in Mtb-infected macrophages, and has widespread effects on innate immune responses. Moreover, they reveal a cascade of AHR-driven innate immune signaling, as IL1B (IL-1β) and IL23 stimulate T cell subsets producing IL22, another direct target of AHR transactivation. Gene expression profiling of Mtb-infected THP-1 monocytic cells following siRNA-mediated Aryl hydrocarbon receptor (AHR) knockdown.

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Human In-vitro Inflammatory Liver Model Recapitulates Immune-Associated Drug Efects with High Predictivity

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