Introduction. Flucloxacillin is a β-lactam antibiotic associated with a high incidence of idiosyncratic drug-induced liver reactions. Although expression of HLA-B*57:01 increases susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the modification of peptides that are presented by the risk allele. In this study, the binding of flucloxacillin was characterized in terminally differentiated liver like HepaRG cells. Here, the membrane transporters involved in drug localisation were investigated. Additionally, due to the immune element of the onset of hypersensitivity, the precise nature of the MHC peptides presented by HLA-B*57:01 were identified.
Methods. Flucloxacillin protein binding was assessed in HepaRG and C1R-B*57:01 cells; B-lymphoblast cells transfected with HLA-B*57:01, using immunofluorescence microscopy. To identify drug modified MHC peptides presented by the risk allele, C1R-B*57:01 cells, used as a model for antigen presentation, were incubated with flucloxacillin for 48h. HLA peptide complexes were subsequently eluted and processed for mass spectrometric analysis.
Results. Direct modification of multiple proteins was observed, which could lead to neo-antigens being presented. Flucloxacillin was shown to disrupt transporter activity in HepaRG cells with localization appearing in bile canaliculi regions. The localization of flucloxacillin was likely mediated primarily by the membrane transporter MRP2. Of the peptides eluted from flucloxacillin treated C1R-B*57:01 cells, 6 peptides were fully annotated to show flucloxacillin-lysine covalent binding. Changes in the overall peptide repertoire were also observed.
Conclusions. We have demonstrated that localization of flucloxacillin occurs in the site of clinical disease during flucloxacillin-induced liver injury. We also demonstrated that neo-antigens, including drug-modified peptides, are presented by HLA-B*57:01. Further investigation into the immunogenicity of haptenated proteins and MHC peptides in the onset of iDILI is ongoing.