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The late event onset of a fraction of idiosyncratic drug-induced liver injury (DILI) cases and the link observed by genome-wide association studies (GWASs) of certain human leucocyte antigen (HLA) alleles with DILI due to specific drugs support the crucial role of the immune system (both innate and adaptive) in the pathogenesis of DILI. Recent advances in both flow and mass cytometry have allowed the profiling of all major immune cell types in a given sample. Therefore, determining the lymphocyte populations in samples from patients with DILI would facilitate the development of specific biomarkers for DILI diagnosis and prognosis. To date, a few studies have explored the immune landscape in DILI. In a recent study of leukocyte immunophenotyping using flow cytometry from the Spanish DILI Registry, an important role of adaptive immune response in DILI is suggested. DILI patients had significantly higher levels of T helper 1 (Th1) cells and activated helper and cytotoxic T cells than healthy controls. Furthermore, the increased expression of negative immune checkpoints and ligands in DILI patients could reflect a restoration of the immune homeostasis. Differences in the profile of cytokines in DILI patients from the Drug-Induced Liver Injury Network (DILIN) also suggest an involvement of both innate and adaptive immune systems in DILI development and prognosis. Moreover, several studies based on immunophenotyping of liver infiltrates showed a distinctive pattern of cellular infiltrates in patients with immune checkpoint inhibitors (ICIs)-DILI, with lower levels of plasma cells, CD20+ B cells and CD4+ T cells than in autoimmune hepatitis (AIH) patients. These pioneering studies highlight the importance of immunophenotyping for the mechanistic understanding of DILI. In this review, available data on immunophenotyping in DILI are gathered, and the potential clinical applications of cutting-edge, novel immunophenotyping techniques are discussed.

Drug-induced liver injury (DILI) can mimic almost all other liver disorders. A phenotype increasingly ascribed to drugs is autoimmune-like hepatitis (ALH). This article summarizes the major topics discussed at a joint International Conference held between Drug-Induced Liver Injury consortium and the International Autoimmune Hepatitis Group. DI-ALH is a liver injury with laboratory and/or histological features that may be indistinguishable from those of autoimmune hepatitis (AIH). Previous studies have revealed that patients with DI-ALH and those with idiopathic AIH have very similar clinical, biochemical, immunological and histological features. Differentiating DI-ALH from AIH is important as patients with DI-ALH rarely require long-term immunosuppression and often resolve spontaneously after stopping the culprit drug whereas patients with AIH mostly need long-term immunosuppression. Therefore, revision of the diagnosis on long-term follow up may be necessary in some cases. More than 40 different drugs including nitrofurantoin, methyldopa, hydralazine, minocycline, infliximab, herbal and dietary supplements such as Khat and Tinospora cordifolia have been implicated in DI-ALH. Understanding of DI-ALH is limited by the lack of specific markers of the disease that could allow a precise diagnosis and similarly, there is no single feature which is diagnostic of AIH. A management algorithm is proposed. There is an urgent need to prospectively evaluate patients with DI-ALH systematically to enable definitive characterization of this condition.

Aims: Detection and characterization of idiosyncratic drug-induced liver injury (DILI) currently rely on standard liver tests, which are suboptimal in terms of specificity, sensitivity and prognosis. Therefore, DILI diagnosis can be delayed, with important consequences for the patient. In this study, we aimed to evaluate the potential of osteopontin, cytokeratin-18 (caspase-cleaved: ccK18 and total: K18), α-glutathione- S-transferase and microRNA-122 as new DILI biomarkers. Methods: Serial blood samples were collected from 32 DILI and 34 non-DILI acute liver injury (ALI) cases and a single sample from 43 population controls without liver injury (HLC) and analysed using enzyme-linked immunosorbent assay (ELISA) or single-molecule arrays. Results: All biomarkers differentiated DILI and ALI from HLC with an area under receiver operator characteristic curve (AUC) value of >0.75 but were less efficient in distinguishing DILI from ALI, with ccK18 (0.79) and K18 (0.76) demonstrating highest potential. However, the AUC improved considerably (0.98) for ccK18 when comparing DILI and a subgroup of autoimmune hepatitis cases. Cytokeratin-18, microRNA- 122 and α-glutathione-S-transferase correlated well with traditional transaminases, while osteopontin correlated most strongly with the international normalized ratio (INR). Conclusions: ccK18 appears promising in distinguishing DILI from autoimmune hepatitis but less so from other forms of acute liver injury. Osteopontin demonstrates prognostic potential with higher levels detected in more severe cases regardless of aetiology, Consejería de Salud y Familia de la Junta de Andalucía, Grant/Award Numbers: PI- 0274-2016, P18-RT-3364, Instituto de Salud Carlos III (ISCIII) cofounded by Fondo Europeo de Desarrollo Regional - FEDER, Grant/Award Numbers: PI19/00883, PI18/00901, UMA18-FEDERJA-193; Universidad de Málaga/CBUA

The identification of specific human leukocyte antigen (HLA) risk alleles in drug-induced liver injury (DILI) points towards an important role of the adaptive immune system in DILI development. In this study we aimed to corroborate the role of an adaptive immune response in DILI through immunophenotyping of leukocyte populations and immune checkpoint expressions. Blood samples were collected from adjudicated DILI (n=12), acute viral hepatitis (VH, 13), acute autoimmune hepatitis (AIH, 9) and acute liver injury of unknown etiology (UKE, 15) at day 1 (recognition), day 7 and day >30. Blood samples from non-alcoholic fatty liver disease patients (NAFLD, 20) and healthy liver controls (HLC, 54) were extracted at one time point. Leukocyte populations and immune checkpoint expressions were determined based on cell surface receptors, except for CTLA-4 that was determined intracellularly, using flow cytometry. At recognition DILI demonstrated significantly higher levels of activated helper T-cell (p

This work was supported in part by a grant from the Instituto de Salud Carlos III (Spain) (PI18/01804, PI19/00883, PI21/01248), from the Consejeria de Economia, Conocimiento, Empresas y Universidad (Junta de Andalucia, Spain) (UMA18-FEDERJA-194, PI18-RT-3364), and from the Consejeria de Salud (Junta de Andalucia, Spain) (PI-0285-2016). This study has been co-funded by FEDER funds ("A way to make Europe") ("Andalucia se mueve con Europa")., Background: Non-alcoholic fatty liver disease (NAFLD) and idiosyncratic drug-induced liver injury (DILI) could share molecular mechanisms involving the immune system. We aimed to identify activation immunological biomarkers in invariant natural killer T (iNKT) and CD4/CD8+ T cells in NAFLD and DILI. Methods: We analyzed the activation profile (CD69, CD25, and HLA-DR) and natural killer group 2 member D (NKG2D) on iNKT cells, and CD4/CD8 T cells in peripheral blood mononuclear cells from NAFLD, with or without significant liver fibrosis, and DILI patients. Results: There was an increase in iNKT cells in NAFLD patients compared to DILI or control subjects. Regarding the cellular activation profile, NAFLD with significant liver fibrosis (F >= 2) displayed higher levels of CD69+iNKT cells compared to NAFLD with none or mild liver fibrosis (F, Instituto de Salud Carlos III PI18/01804 PI19/00883 PI21/01248, Junta de Andalucia UMA18-FEDERJA-194 PI18-RT-3364 PI-0285-2016, FEDER funds ("A way to make Europe") ("Andalucia se mueve con Europa")

Introduction: Idiosyncratic DILI is a challenging condition, believed to involve the immune system. This hypothesis is supported by various identified HLA risk alleles. Objectives: To evaluate a potential role of the immune system in DILI through leukocyte immunophenotyping. Methods: Blood samples were collected from adjudicated DILI (n=12) and viral hepatitis (VH, 13) at day 1 (recognition), 7 and >30. A single blood sample was extracted from healthy liver controls (HLC, 54). Leukocyte populations and immune checkpoint expressions were determined based on cell surface receptors, except for CTLA-4 that was determined intracellularly, using multiparametric flow cytometry. Results: No differences were detected in leukocytes, lymphocytes or neutrophils counts at day 1. However, DILI (0.57 × 10E09/L, p=0.037) and HV (1.41 × 10E09/L, p

The gut microbiota could play a significant role in the progression of nonalcoholic fatty liver disease (NAFLD); however, its relevance in drug-induced liver injury (DILI) remains unexplored. Since the two hepatic disorders may share damage pathways, we analysed the metagenomic profile of the gut microbiota in NAFLD, with or without significant liver fibrosis, and in DILI, and we identified the main associated bacterial metabolic pathways. In the NAFLD group, we found a decrease in Alistipes, Barnesiella, Eisenbergiella, Flavonifractor, Fusicatenibacter, Gemminger, Intestinimonas, Oscillibacter, Parasutterella, Saccharoferementans and Subdoligranulum abundances compared with those in both the DILI and control groups. Additionally, we detected an increase in Enterobacter, Klebsiella, Sarcina and Turicibacter abundances in NAFLD, with significant liver fibrosis, compared with those in NAFLD with no/mild liver fibrosis. The DILI group exhibited a lower microbial bacterial richness than the control group, and lower abundances of Acetobacteroides, Blautia, Caloramator, Coprococcus, Flavobacterium, Lachnospira, Natronincola, Oscillospira, Pseudobutyrivibrio, Shuttleworthia, Themicanus and Turicibacter compared with those in the NAFLD and control groups. We found seven bacterial metabolic pathways that were impaired only in DILI, most of which were associated with metabolic biosynthesis. In the NAFLD group, most of the differences in the bacterial metabolic pathways found in relation to those in the DILI and control groups were related to fatty acid and lipid biosynthesis. In conclusion, we identified a distinct bacterial profile with specific bacterial metabolic pathways for each type of liver disorder studied. These differences can provide further insight into the physiopathology and development of NAFLD and DILI. This work was supported in part by a grant from the Instituto de Salud Carlos III (Spain) (PI18/01804, PI19/00883, PI21/01248), from the Consejería de Economía, Conocimiento, Empresas y Universidad (Junta de Andalucía, Spain) (PI18–RT‐3364, UMA18-FEDERJA-194), and from the Consejería de Salud (Junta de Andalucía, Spain) (PI-0285–2016). This study has been co-funded by FEDER funds (“A way to make Europe”) (“Andalucía se mueve con Europa”). CRD is supported by a grant from the Consejería de Transformación Económica, Industria, Conocimiento y Universidades de Junta de Andalucía (Spain) (DOC_01610). FMR is supported by a grant from the ISCIII (Spain) (FI19/00189). AC is supported by a grant from the ISCIII (Spain) (IFI18/00047). EGF is supported by the Nicolas Monardes program from the Consejería de Salud de Andalucía (Spain) (C-0031–2016). Funding for open access charge: Universidad de Málaga / CBUA (Spain).

Introduction: Idiosyncratic drug-induced liver injury (DILI) is a complex hepatic condition whose diagnosis is challenging due to lack of specific biomarkers. Objectives: We aimed to evaluate serum metabolomic differences between patients with DILI and with other causes of liver injury in search for specific DILI biomarkers. Methods: Metabolomic profiles of serum samples from 26 Spanish DILI patients, 34 with non-DILI acute liver injury (ALI) and 48 healthy controls, were analyzed using UHPLC-MS. To assess changes in disease progression, DILI and ALI patients were followed-up from detection (visit 1), one week (visit 2) and >30 days (visit 3). Data were analyzed using Shapiro-Wilk, Student's t and Wilcoxon tests. Results: Several amino acids, fatty acids (FAs), LPI and bile acids were increased, whereas the glycerophospholipids MEPE and MAPC were decreased (p0,05). Conclusion: Most metabolomic differences are found at times closer to DILI recognition (visits 1&2), although abnormal values of FAs remain during recovery. Some FAs species and the amino acids taurine, Phe-Phe glutamic acid, lysine, tryptophan and alanine seem promising DILI biomarker candidates that should be further explored. Funding: CIBERehd, ISCiii-FEDER PI18/00901, PI19/00883.