Final Report Summary - CHIC-FILI (Definition of the chemical and immunological characteristics of flucloxacillin-induced liver injury)
Drug-induced liver injury (DILI) is a major concern for public health as it is a severe and common form of adverse drug reaction. Furthermore, reactions have a socio-economic impact as they are a leading cause of drug withdrawal.
The effective combination of amoxicillin and clavulanic acid (co-amoxiclav) provides broad-spectrum anti-microbial activity, which makes it one of the most frequently prescribed antibiotics throughout the world. Most individuals safely tolerate the drug; however, a small portion patients report severe and occasionally life-threatening liver injury. Several genetic studies have shown that individual susceptibility to co-amoxiclav induced liver injury is linked to a specific HLA haplotype, HLA-DRB1*1501-DQB1*0602 (Hautekeete ML et al. 1999 Gastroenterology 117: 1181–1186, Lucena MI et al. 2011 Gastroenterology 141: 338–347).
Although co-amoxiclav induced liver injury is a well-recognized complication both in clinics and pharmacogenomics, the underlying immune mechanism is still poorly understood. Here, we examined drug-specific lymphocyte proliferation from patients with co-amoxiclav induced liver injury and investigated the HLA-restricted T-cell immune response to the drug combination using T-cell clones.
Peripheral blood mononuclear cells from 7 patients were cultured in vitro with amoxicillin, clavulanic acid, or co-amoxiclav. All patients showed a strong lymphocyte proliferative response to amoxicillin. Increased interferon γ (IFN-γ) secretion was also found in the presence of amoxicillin using ELISpot as a readout for the antigen-specific T-cell response. Interestingly, there was no proliferative response to clavulanic acid. However, IFN-γ secretion was enhanced. Optimal T-cell stimulation was observed for both drugs in the range of 0.5mM to 2mM.
T-cell clones against amoxicillin and clavulanic acid were generated from 3 patients by limiting dilution. Amoxicillin-specific T-cell clones showed a strong proliferative response and IFN-γ secretion to amoxicillin and in a dose dependent manner. Clavulanic acid-specific T-cell clones proliferated weakly in the presence of the drug, but high levels of IFN-γ secretion was observed (in the presence of clavulanic acid and co-amoxiclav). Importantly, amoxicillin and clavulanic acid-specific T-cell clones were not activated in the presence of the alternative drug-antigen (i.e. amoxicillin-responsive T-cell clones specifically recognized amoxicillin, but not clavulanate, and vice versa). Phenotype analysis using flow cytometry demonstrated that all T-cell clones had CD4+ /CD45RO+.
HLA-restricted T-cell proliferation was examined using amoxicillin and clavulanic acid-specific T-cell clones and anti-MHC blocking antibodies. The proliferative response of the T-cell clones was strongly inhibited by an anti-HLA (anti-HLA-DR) class II antibody. IFN-γ secretion was also strongly inhibited in the presence of the anti-HLA-DR antibody. The amoxicillin-responsive clones were then used to define the HLA-DR allele(s) that the drug interacts with to stimulate a T-cell response. Amoxicillin-responsive clones were stimulated to proliferate with allogenic antigen-presenting cells expressing HLA-DRB1*1501. In contrast, antigen-presenting cells expressing HLA-DRB1*0701 didn’t stimulate the clones. IFN-γ secretion from amoxicillin-specific T-cell clones showed the same HLA-restriction. These data demonstrate that the drug-specific T-cell response is dependent on the drug-derived antigen interacting with specific MHC class II molecules.
To study the priming of naïve T-cells from drug-naïve donors, we utilized our recently established dendritic cell/T-cell co-culture system (Faulkner et al., 2012 Toxicol Sci. 127:150-8). T-cells from a HLA-DRB1*1501 positive donor were effectively primed against amoxicillin when dendritic cells were used to present the drug-derived antigen. Following amoxicillin restimulation, the primed T-cells were found to proliferate and secrete cytokines such as IFN-γ and IL-13.
To examine cross-reactivity of the amoxicillin-specific T-cells, clones were cultured with autologous APC and alternative ß–lactam antibiotics such as piperacillin, penicillin G, and flucloxacillin which modify similar lysine residues on albumin. These experiments revealed that the T-cell response was highly drug specific; clones were activated with amoxicillin, but not the other structurally-related drugs.
Mass spectrometric techniques were used to define the chemistry of drug–protein conjugation using human serum albumin as a model target. Amoxicillin and clavulanic acid were found to bind in a time- and concentration-dependent fashion to specific lysine residues.
Collectively, these findings suggest that a major susceptibility factor relates to the restriction of the fit of the antigen into particular immunological receptors in co-amoxiclav induced liver injury and the HLA-DRB1*1501 risk genotype relates the drug-specific immune response. Based on this study, we have designed a prospective clinical study to evaluate the HLA genotype on pharmacokinetics/pharmacodynamics after multiple administration of co-amoxiclav and explore representative biomarkers for drug-induced liver injury in Korean normal healthy controls.
Socio-economic impact: There are no available methods to diagnose immune-mediated drug reactions in patients with drug-induced liver injury. This project indicates that in vitro assays characterizing drug-antigen-specific T-cells can be used to diagnose immunological drug-induced liver injury. This experimental approach can be applied to drug screening for candidate drugs and assist patient management by warning usage in susceptible patients.
The effective combination of amoxicillin and clavulanic acid (co-amoxiclav) provides broad-spectrum anti-microbial activity, which makes it one of the most frequently prescribed antibiotics throughout the world. Most individuals safely tolerate the drug; however, a small portion patients report severe and occasionally life-threatening liver injury. Several genetic studies have shown that individual susceptibility to co-amoxiclav induced liver injury is linked to a specific HLA haplotype, HLA-DRB1*1501-DQB1*0602 (Hautekeete ML et al. 1999 Gastroenterology 117: 1181–1186, Lucena MI et al. 2011 Gastroenterology 141: 338–347).
Although co-amoxiclav induced liver injury is a well-recognized complication both in clinics and pharmacogenomics, the underlying immune mechanism is still poorly understood. Here, we examined drug-specific lymphocyte proliferation from patients with co-amoxiclav induced liver injury and investigated the HLA-restricted T-cell immune response to the drug combination using T-cell clones.
Peripheral blood mononuclear cells from 7 patients were cultured in vitro with amoxicillin, clavulanic acid, or co-amoxiclav. All patients showed a strong lymphocyte proliferative response to amoxicillin. Increased interferon γ (IFN-γ) secretion was also found in the presence of amoxicillin using ELISpot as a readout for the antigen-specific T-cell response. Interestingly, there was no proliferative response to clavulanic acid. However, IFN-γ secretion was enhanced. Optimal T-cell stimulation was observed for both drugs in the range of 0.5mM to 2mM.
T-cell clones against amoxicillin and clavulanic acid were generated from 3 patients by limiting dilution. Amoxicillin-specific T-cell clones showed a strong proliferative response and IFN-γ secretion to amoxicillin and in a dose dependent manner. Clavulanic acid-specific T-cell clones proliferated weakly in the presence of the drug, but high levels of IFN-γ secretion was observed (in the presence of clavulanic acid and co-amoxiclav). Importantly, amoxicillin and clavulanic acid-specific T-cell clones were not activated in the presence of the alternative drug-antigen (i.e. amoxicillin-responsive T-cell clones specifically recognized amoxicillin, but not clavulanate, and vice versa). Phenotype analysis using flow cytometry demonstrated that all T-cell clones had CD4+ /CD45RO+.
HLA-restricted T-cell proliferation was examined using amoxicillin and clavulanic acid-specific T-cell clones and anti-MHC blocking antibodies. The proliferative response of the T-cell clones was strongly inhibited by an anti-HLA (anti-HLA-DR) class II antibody. IFN-γ secretion was also strongly inhibited in the presence of the anti-HLA-DR antibody. The amoxicillin-responsive clones were then used to define the HLA-DR allele(s) that the drug interacts with to stimulate a T-cell response. Amoxicillin-responsive clones were stimulated to proliferate with allogenic antigen-presenting cells expressing HLA-DRB1*1501. In contrast, antigen-presenting cells expressing HLA-DRB1*0701 didn’t stimulate the clones. IFN-γ secretion from amoxicillin-specific T-cell clones showed the same HLA-restriction. These data demonstrate that the drug-specific T-cell response is dependent on the drug-derived antigen interacting with specific MHC class II molecules.
To study the priming of naïve T-cells from drug-naïve donors, we utilized our recently established dendritic cell/T-cell co-culture system (Faulkner et al., 2012 Toxicol Sci. 127:150-8). T-cells from a HLA-DRB1*1501 positive donor were effectively primed against amoxicillin when dendritic cells were used to present the drug-derived antigen. Following amoxicillin restimulation, the primed T-cells were found to proliferate and secrete cytokines such as IFN-γ and IL-13.
To examine cross-reactivity of the amoxicillin-specific T-cells, clones were cultured with autologous APC and alternative ß–lactam antibiotics such as piperacillin, penicillin G, and flucloxacillin which modify similar lysine residues on albumin. These experiments revealed that the T-cell response was highly drug specific; clones were activated with amoxicillin, but not the other structurally-related drugs.
Mass spectrometric techniques were used to define the chemistry of drug–protein conjugation using human serum albumin as a model target. Amoxicillin and clavulanic acid were found to bind in a time- and concentration-dependent fashion to specific lysine residues.
Collectively, these findings suggest that a major susceptibility factor relates to the restriction of the fit of the antigen into particular immunological receptors in co-amoxiclav induced liver injury and the HLA-DRB1*1501 risk genotype relates the drug-specific immune response. Based on this study, we have designed a prospective clinical study to evaluate the HLA genotype on pharmacokinetics/pharmacodynamics after multiple administration of co-amoxiclav and explore representative biomarkers for drug-induced liver injury in Korean normal healthy controls.
Socio-economic impact: There are no available methods to diagnose immune-mediated drug reactions in patients with drug-induced liver injury. This project indicates that in vitro assays characterizing drug-antigen-specific T-cells can be used to diagnose immunological drug-induced liver injury. This experimental approach can be applied to drug screening for candidate drugs and assist patient management by warning usage in susceptible patients.