The use of PD-1 functional knockout rats to study idiosyncratic adverse reactions to nevirapine.

Idiosyncratic drug reactions (IDRs) are associated with significant patient morbidity/mortality and lead to considerable drug candidate attrition in drug development. Their idiosyncratic nature makes the study of IDRs difficult. In particular, nevirapine is associated with a relatively high risk of serious skin rash and liver injury. We previously found that nevirapine causes a similar skin rash in female Brown Norway rats, but these animals do not develop significant liver injury. Programmed cell death protein-1 (PD-1) is an immune checkpoint involved in immune tolerance, and anti-PD-1 antibodies have been used to treat cancer. However, they increase the risk of liver injury caused by co-administered drugs. We found that PD-1-/- mice are more susceptible to drug-induced liver injury, but PD-1-/- mice are not a good model for all drugs. In particular, they do not develop a skin rash when treated with nevirapine, at least in part because they lack the sulfotransferase in their skin that forms the reactive metabolite responsible for the rash. Therefore, we developed a PD-1 mutant (PD-1m/m) rat, with an excision in the ligand-binding domain of PD-1, to test whether nevirapine would cause a more serious skin rash in these animals. The PD-1m/m rat was based on a Sprague Dawley background, which has a lower incidence of skin rash than Brown Norway rats. The treated PD-1m/m rats developed more severe liver injury than PD-1-/- mice, but in contrast to expectations, they did not develop a skin rash. Functional knockouts provide a unique tool to study the mechanisms of IDRs.

Genome-wide CRISPR screens identify novel regulators of wild-type and mutant p53 stability.

Tumor suppressor p53 (TP53) is frequently mutated in cancer, often resulting not only in loss of its tumor-suppressive function but also acquisition of dominant-negative and even oncogenic gain-of-function traits. While wild-type p53 levels are tightly regulated, mutants are typically stabilized in tumors, which is crucial for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. Most regulators of wild-type p53 also regulate p53 mutants, except for p53 R337H regulators, which are largely private to this mutant. Mechanistically, FBXO42 emerged as a positive regulator for a subset of p53 mutants, working with CCDC6 to control USP28-mediated mutant p53 stabilization. Additionally, C16orf72/HAPSTR1 negatively regulates both wild-type p53 and all tested mutants. C16orf72/HAPSTR1 is commonly amplified in breast cancer, and its overexpression reduces p53 levels in mouse mammary epithelium leading to accelerated breast cancer. This study offers a network perspective on p53 stability regulation, potentially guiding strategies to reinforce wild-type p53 or target mutant p53 in cancer.

NFATC2IP is a mediator of SUMO-dependent genome integrity.

The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to genome duplication and repair. To investigate the mechanisms underpinning the essential function of SUMO, we undertook a genome-scale CRISPR/Cas9 screen probing the response to SUMOylation inhibition. This effort identified 130 genes whose disruption reduces or enhances the toxicity of TAK-981, a clinical-stage inhibitor of the SUMO E1-activating enzyme. Among the strongest hits, we validated and characterized NFATC2IP, an evolutionarily conserved protein related to the fungal Esc2 and Rad60 proteins that harbors tandem SUMO-like domains. Cells lacking NFATC2IP are viable but are hypersensitive to SUMO E1 inhibition, likely due to the accumulation of mitotic chromosome bridges and micronuclei. NFATC2IP primarily acts in interphase and associates with nascent DNA, suggesting a role in the postreplicative resolution of replication or recombination intermediates. Mechanistically, NFATC2IP interacts with the SMC5/6 complex and UBC9, the SUMO E2, via its first and second SUMO-like domains, respectively. AlphaFold-Multimer modeling suggests that NFATC2IP positions and activates the UBC9-NSMCE2 complex, the SUMO E3 ligase associated with SMC5/SMC6. We conclude that NFATC2IP is a key mediator of SUMO-dependent genomic integrity that collaborates with the SMC5/6 complex.

The development and characterization of a CRISPR/Cas9-mediated PD-1 functional knockout rat as a tool to study idiosyncratic drug reactions.

Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine.

The Putative Drosophila TMEM184B Ortholog Tmep Ensures Proper Locomotion by Restraining Ectopic Firing at the Neuromuscular Junction.

TMEM184B is a putative seven-pass membrane protein that promotes axon degeneration after injury. TMEM184B mutation causes aberrant neuromuscular architecture and sensory and motor behavioral defects in mice. The mechanism through which TMEM184B causes neuromuscular defects is unknown. We employed Drosophila melanogaster to investigate the function of the closely related gene, Tmep (CG12004), at the neuromuscular junction. We show that Tmep is required for full adult viability and efficient larval locomotion. Tmep mutant larvae have a reduced body contraction rate compared to controls, with stronger deficits in females. In recordings from body wall muscles, Tmep mutants show substantial hyperexcitability, with many postsynaptic potentials fired in response to a single stimulation, consistent with a role for Tmep in restraining synaptic excitability. Additional branches and satellite boutons at Tmep mutant neuromuscular junctions are consistent with an activity-dependent synaptic overgrowth. Tmep is expressed in endosomes and synaptic vesicles within motor neurons, suggesting a possible role in synaptic membrane trafficking. Using RNAi knockdown, we show that Tmep is required in motor neurons for proper larval locomotion and excitability, and that its reduction increases levels of presynaptic calcium. Locomotor defects can be rescued by presynaptic knockdown of endoplasmic reticulum calcium channels or by reducing evoked release probability, further suggesting that excess synaptic activity drives behavioral deficiencies. Our work establishes a critical function for Tmep in the regulation of synaptic transmission and locomotor behavior.

Dissecting a disynaptic central amygdala-parasubthalamic nucleus neural circuit that mediates cholecystokinin-induced eating suppression.

OBJECTIVE: Cholecystokinin (CCK) plays a critical role in regulating eating and metabolism. Previous studies have mapped a multi-synapse neural pathway from the vagus nerve to the central nucleus of the amygdala (CEA) that mediates the anorexigenic effect of CCK. However, the neural circuit downstream of the CEA is still unknown due to the complexity of the neurons in the CEA. Here we sought to determine this circuit using a novel approach. METHODS: It has been established that a specific population of CEA neurons, marked by protein kinase C-delta (PKC-δ), mediates the anorexigenic effect of CCK by inhibiting other CEA inhibitory neurons. Taking advantage of this circuit, we dissected the neural circuit using a unique approach based on the idea that neurons downstream of the CEA should be disinhibited by CEAPKC-δ+ neurons while being activated by CCK. We also used optogenetic assisted electrophysiology circuit mapping and in vivo chemogenetic manipulation methods to determine the circuit structure and function. RESULTS: We found that neurons in the parasubthalamic nucleus (PSTh) are activated by the activation of CEAPKC-δ+ neurons and by the peripheral administration of CCK. We demonstrated that CEAPKC-δ+ neurons inhibit the PSTh-projecting CEA neurons; accordingly, the PSTh neurons can be disynaptically disinhibited or "activated" by CEAPKC-δ+ neurons. Finally, we showed that chemogenetic silencing of the PSTh neurons effectively attenuates the eating suppression induced by CCK. CONCLUSIONS: Our results identified a disynaptic CEA-PSTh neural circuit that mediates the anorexigenic effect of CCK and thus provide an important neural mechanism of how CCK suppresses eating.

Transmembrane protein TMEM184B is necessary for interleukin-31-induced itch.

ABSTRACT: Nociceptive and pruriceptive neurons in the dorsal root ganglia (DRG) convey sensations of pain and itch to the spinal cord, respectively. One subtype of mature DRG neurons, comprising 6% to 8% of neurons in the ganglia, is responsible for sensing mediators of acute itch and atopic dermatitis, including the cytokine IL-31. How itch-sensitive (pruriceptive) neurons are specified is unclear. Here, we show that transmembrane protein 184B (TMEM184B), a protein with roles in axon degeneration and nerve terminal maintenance, is required for the expression of a large cohort of itch receptors, including those for interleukin 31 (IL-31), leukotriene C4, and histamine. Male and female mice lacking TMEM184B show reduced responses to IL-31 but maintain normal responses to pain and mechanical force, indicating a specific behavioral defect in IL-31-induced pruriception. Calcium imaging experiments indicate that a reduction in IL-31-induced calcium entry is a likely contributor to this phenotype. We identified an early failure of proper Wnt-dependent transcriptional signatures and signaling components in Tmem184b mutant mice that may explain the improper DRG neuronal subtype specification. Accordingly, lentiviral re-expression of TMEM184B in mutant embryonic neurons restores Wnt signatures. Together, these data demonstrate that TMEM184B promotes adult somatosensation through developmental Wnt signaling and promotion of proper pruriceptive gene expression. Our data illuminate a new key regulatory step in the processes controlling the establishment of diversity in the somatosensory system.

Artificial intelligence for the discovery of novel antimicrobial agents for emerging infectious diseases.

The search for effective drugs to treat new and existing diseases is a laborious one requiring a large investment of capital, resources, and time. The coronavirus 2019 (COVID-19) pandemic has been a painful reminder of the lack of development of new antimicrobial agents to treat emerging infectious diseases. Artificial intelligence (AI) and other in silico techniques can drive a more efficient, cost-friendly approach to drug discovery by helping move potential candidates with better clinical tolerance forward in the pipeline. Several research teams have developed successful AI platforms for hit identification, lead generation, and lead optimization. In this review, we investigate the technologies at the forefront of spearheading an AI revolution in drug discovery and pharmaceutical sciences.

Testing Possible Risk Factors for Idiosyncratic Drug-Induced Liver Injury Using an Amodiaquine Mouse Model and Co-treatment with 1-Methyl-d-Tryptophan or Acetaminophen.

Idiosyncratic drug reactions are unpredictable adverse reactions. Although most such adverse reactions appear to be immune mediated, their exact mechanism(s) remain elusive. The idiosyncratic drug reaction most associated with serious consequences is idiosyncratic drug-induced liver injury (IDILI). We have developed a mouse model of amodiaquine (AQ)-induced liver injury that reflects the clinical characteristics of IDILI in humans. This was accomplished by impairing immune tolerance by using PD-1-/- mice and an antibody against CTLA-4. PD-1 and CTLA-4 are known negative regulators of lymphocyte activation, which promote immune tolerance. Immune checkpoint inhibitors have become important tools for the treatment of cancer. However, as in our model, immune checkpoint inhibitors increase the risk of IDILI with drugs that have an incidence of causing liver injury. Agents such as 1-methyl-d-tryptophan (D-1-MT), an inhibitor of the immunosuppressive indoleamine 2,3-dioxygenase (IDO) enzyme, have also been proposed as anti-cancer treatments. Another possible risk factor for the induction of an immune response is the release of danger-associated molecular patterns (DAMPs). Acetaminophen (APAP) is known to cause acute liver injury, and it is likely to cause the release of DAMPs. Therefore, either of these agents could increase the risk of IDILI, although through different mechanisms. If true, then this would have clinical implications. We found that co-treatment with D-1-MT paradoxically decreased liver injury in our model, and although APAP appeared to slightly increase AQ-induced liver injury, the difference was not significant. Such results highlight the complexity of the immune response, which makes potential interactions difficult to predict.

Liver Injury Caused by Green Tea Extract in PD-1-/- Mice: An Impaired Immune Tolerance Model for Idiosyncratic Drug-Induced Liver Injury.

Idiosyncratic drug-induced liver injury (IDILI) is an idiosyncratic drug reaction that is specific to an individual and can lead to liver failure and even death. The mechanism of IDILI remains poorly understood, but most IDILI appears to be immune-mediated. We have developed the first validated animal model by using a PD-1-/- mouse model in combination with anti-CTLA-4 to block immune checkpoints and impair immune tolerance. Treatment of these mice with drugs that cause IDILI in humans led to delayed-onset liver injury with characteristics similar to IDILI in humans. The current study investigates the effects of green tea extract, a weight-loss dietary supplement that has been reported to cause IDILI in humans. Green tea extracts contain a highly variable content of catechins including (-)-epigallocatechin gallate, the major catechin in green tea formulations. If the liver injury caused by green tea extract in humans is immune-mediated, it may occur in our impaired immune tolerance model. Female PD-1-/- mice treated with anti-CTLA-4 antibody and green tea extract (500 mg/kg), a dose that is considered a no-observed-adverse-effect level for liver in rodents, produced a delayed onset increase in serum alanine transaminase levels and an increase in hepatic CD8+ T cells. In contrast, the response in male PD-1-/- mice was less pronounced, and there was no evidence of liver injury in wild-type mice. These findings are consistent with the hypothesis that the IDILI caused by green tea extract is immune-mediated and is similar to IDILI caused by medications that are associated with IDILI.

A Genetic Map of the Response to DNA Damage in Human Cells.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

Endogenous DNA 3' Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

A consensus set of genetic vulnerabilities to ATR inhibition.

The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase ε accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity and the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.

A bed nucleus of stria terminalis microcircuit regulating inflammation-associated modulation of feeding.

Loss of appetite or anorexia associated with inflammation impairs quality of life and increases morbidity in many diseases. However, the exact neural mechanism that mediates inflammation-associated anorexia is still poorly understood. Here we identified a population of neurons, marked by the expression of protein kinase C-delta, in the oval region of the bed nucleus of the stria terminalis (BNST), which are activated by various inflammatory signals. Silencing of these neurons attenuates the anorexia caused by these inflammatory signals. Our results demonstrate that these neurons mediate bidirectional control of general feeding behaviors. These neurons inhibit the lateral hypothalamus-projecting neurons in the ventrolateral part of BNST to regulate feeding, receive inputs from the canonical feeding regions of arcuate nucleus and parabrachial nucleus. Our data therefore define a BNST microcircuit that might coordinate canonical feeding centers to regulate food intake, which could offer therapeutic targets for feeding-related diseases such as anorexia and obesity.

Rotenone Increases Isoniazid Toxicity but Does Not Cause Significant Liver Injury: Implications for the Hypothesis that Inhibition of the Mitochondrial Electron Transport Chain Is a Common Mechanism of Idiosyncratic Drug-Induced Liver Injury.

Idiosyncratic drug reactions (IDRs) significantly increase the risk of failure in drug development. The major IDR leading to drug candidate failure is idiosyncratic drug-induced liver injury (IDILI). Although most evidence suggests that IDRs are mediated by the immune system, there are other hypotheses, such as mitochondrial dysfunction. Many pharmaceutical companies routinely screen for mitochondrial toxicity in an attempt to "derisk" drug candidates. However, the basic hypothesis has never been rigorously tested. A major assay used for this screening involves measurement of inhibition of the mitochondrial electron transport chain. One study found that the combination of rotenone and isoniazid, which inhibit mitochondrial complex I and II, respectively, were synergistic in causing hepatocyte toxicity in vitro and suggested the combination of another drug that inhibited complex I would increase the risk of isoniazid-induced liver injury in patients. We tested this hypothesis in vivo where wild-type and PD-1-/- mice administered anti-CTLA-4, our impaired immune tolerance mouse model, were given 0.02% (w/v) rotenone in water or 0.1%, 0.05%, and 0.01% (w/w) rotenone alone or in combination with isoniazid in food. The cotreatment led to lethality in 100% of the animals receiving 0.1% rotenone and 0.2% isoniazid and 83% of the animals cotreated with 0.05% rotenone and 0.2% isoniazid in food. Nevertheless, there was no significant increase in GLDH or histological evidence of liver injury. No signs of toxicity were observed in any of the mice given rotenone or isoniazid alone. Even though inhibition of the mitochondrial electron transport chain did not lead to significant liver toxicity, it could provide danger signals that promote immune-mediated liver injury. However, rotenone did not significantly increase the liver injury induced by isoniazid in our impaired immune tolerance model. Overall, we conclude that inhibition of the mitochondrial electron transport chain is not a significant mechanism of IDILI.

3-Methylcholanthrene Induces Chylous Ascites in TCDD-Inducible Poly-ADP-Ribose Polymerase (Tiparp) Knockout Mice.

TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an aryl hydrocarbon receptor (AHR) target gene that functions as part of a negative feedback loop to repress AHR activity. Tiparp-/- mice exhibit increased sensitivity to the toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including lethal wasting syndrome. However, it is not known whether Tiparp-/- mice also exhibit increased sensitivity to other AHR ligands. In this study, we treated male Tiparp-/- or wild type (WT) mice with a single injection of 100 mg/kg 3-methylcholanthrene (3MC). Consistent with TIPARP's role as a repressor of AHR signaling, 3MC-treated Tiparp-/- mice exhibited increased hepatic Cyp1a1 and Cyp1b1 levels compared with WT mice. No 3MC-treated Tiparp-/- mice survived beyond day 16 and the mice exhibited chylous ascites characterized by an accumulation of fluid in the peritoneal cavity. All WT mice survived the 30-day treatment and showed no signs of fluid accumulation. Treated Tiparp-/- mice also exhibited a transient and mild hepatotoxicity with inflammation. 3MC-treated WT, but not Tiparp-/- mice, developed mild hepatic steatosis. Lipid deposits accumulated on the surface of the liver and other abdominal organs in the 3MC-Tiparp-/- mice. Our study reveals that Tiparp-/- mice have increased sensitivity to 3MC-induced liver toxicity, but unlike with TCDD, lethality is due to chylous ascites rather than wasting syndrome.

Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity.

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

Hepatocyte-Specific Deletion of TIPARP, a Negative Regulator of the Aryl Hydrocarbon Receptor, Is Sufficient to Increase Sensitivity to Dioxin-Induced Wasting Syndrome.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparpfl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparpfl/flCreAlb) and whole-body (Tiparpfl/flCreCMV; TiparpEx3-/-) Tiparp null mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice given a single injection of 10 µg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparpfl/flCreAlb and TiparpEx3-/- mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparpfl/flCreAlb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.

The Effects of Immune Modulators on Amodiaquine-Induced Liver Injury.

If idiosyncratic drug-induced liver injury (IDILI) is immune mediated, then it is logical that immune modulators may be able to affect liver injury caused by a drug. We have previously shown that modulating the immune system by impairing programmed cell death protein (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) signaling, both receptors involved in immune tolerance, was capable of producing an animal model of amodiaquine (AQ) IDILI with characteristics very similar to IDILI in humans. Other immune modulators may also increase liver injury caused by drugs that cause IDILI in humans. In this study, myeloid derived suppressor cells (MDSCs), transforming growth factor beta (TGF-ß), and lymphocyte-activation gene 3 (LAG3) were targeted with antibodies, with and without PD-1 and CTLA-4 impairment. We found that anti-Gr1 antibodies used to deplete MDSCs led to a significant increase in AQ-induced liver injury in wild-type mice; however, the injury was actually less in PD-1-/- mice, with or without anti-CTLA-4, and it was less than we have previously observed in PD-1-/- mice combined with anti-CTLA-4 without anti-Gr1. Addition of anti-LAG3 or anti-TGF-ß antibodies produced a small increase ALT in AQ-treated wild-type mice. There was a significant increase in ALT in PD-1-/- mice co-treated with anti-LAG3 or anti-TGF-ß relative to AQ-treated wild-type mice. In the case of TGF-ß, this was further increased by the addition of anti-CTLA-4, but if anything, there appeared to be a paradoxical decrease when anti-CTLA-4 was combined with anti-LAG3. Overall, the results from this study were not always as expected, and they highlight the complexity of the immune response, in particular immune tolerance, which appears to be the dominant immune response to drugs that cause IDILI.