HLA-B*57:01-dependent intracellular stress in keratinocytes triggers dermal hypersensitivity reactions to abacavir.

Specific human leukocyte antigen (HLA) polymorphisms combined with certain drug administration strongly correlate with skin eruption. Abacavir hypersensitivity (AHS), which is strongly associated with HLA-B*57:01, is one of the most representative examples. Conventionally, HLA transmits immunological signals via interactions with T cell receptors on the cell surface. This study focused on HLA-mediated intracellular reactions in keratinocytes that might determine the onset of skin immunotoxicity by drug treatments. Abacavir exposure resulted in keratinocytes expressing HLA-B*57:01 exhibiting endoplasmic reticulum (ER) stress responses, such as immediate calcium release into the cytosol and enhanced HSP70 expression. In contrast, keratinocytes expressing HLA-B*57:03 (closely related to HLA-B*57:01) did not show these changes. This indicated that HLA-B*57:01 has a specific intracellular response to abacavir in keratinocytes in the absence of lymphocytes. Furthermore, abacavir exposure in HLA-B*57:01-expressing keratinocytes elevated the expression of cytokines/chemokines such as interferon-γ, interleukin-1ß, and CCL27, and induced T lymphoblast migration. These effects were suppressed by ER stress relief using 4-phenylbutyrate (4-PB). HLA-B*57:01-transgenic mice also exhibited ER stress in epidermal areas following abacavir administration, and abacavir-induced skin toxicity was attenuated by the administration of 4-PB. Moreover, abacavir bound to HLA-B*57:01 within cells and its exposure led to HLA-B*57:01 protein aggregation and interaction with molecular chaperones in the ER of keratinocytes. Our results underscore the importance of HLA-mediated intracellular stress responses in understanding the onset of HLA-B*57:01-mediated AHS. We provide the possibility that the intracellular behavior of HLA is crucial for determining the onset of drug eruptions.

Pathological changes in various organs in HLA-B*57:01 transgenic mice with abacavir-induced skin eruption.

Several patients with cutaneous adverse drug reactions exhibit extracutaneous organ damages, and it becomes severe in a few patients resulting in death due to multiorgan failure. Understanding the sequential changes in various organs in patients with cutaneous eruption following drug administration will help understand disease onset and progression, aiding the development of prevention strategies and interventions. Therefore, we aimed to understand the effects of abacavir (ABC) on various organs in patients with ABC-induced eruptions by evaluating its effects in a mouse model. We found pathological changes in various organs of HLA-B*57:01 transgenic mice (B*57:01-Tg) following oral administration of ABC (20 mg/body/day). B*57:01-Tg exhibited a significant body weight decrease from day 1 of ABC administration, and reddening of the auricle was observed from day 5, and approximately 2/3 mice died by day 7. Histopathological examination revealed severe thymic atrophy after day 3, infiltration of inflammatory cells, predominantly lymphocytes with neutrophils, not only in the skin but also in the liver, kidney, and lung after day 5, and an increased number of lymphocytes with enlarged nuclei and granulocytic hematopoiesis were observed in the spleen after day 5. Blood chemistry revealed that albumin/globulin ratio was below 1.0 on day 5, reflecting a systemic inflammatory response, and the aspartate aminotransferase concentration rose to 193 ± 93.0 U/L on day 7, suggesting that cell damage may have occurred in various organs including liver accompanying inflammatory cell infiltration. These examinations of a mouse model of ABC-induced skin eruption show that disorders in various organs other than the skin should be considered and provide insights into the unexpected early systemic responses dependent on HLA-B*57:01. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00220-1.

siRNA delivery to lymphatic endothelial cells via ApoE-mediated uptake by lipid nanoparticles.

Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.

TARC/CCL17 Expression Is Associated with CD8+ T Cell Recruitment in Abacavir-Induced Skin Hypersensitivity in HLA-Transgenic Mice.

Abacavir (ABC)-induced hypersensitivity (AHS) is strongly associated with human leukocyte antigen (HLA)-B*57 : 01 expression. Previous studies have demonstrated the feasibility of applying the HLA-transgenic mouse model in this context. ABC-induced adverse reactions were observed in HLA-B*57 : 01 transgenic (B*57 : 01-Tg) mice. Moreover, regulating immune tolerance could result in severe AHS that mimics symptoms observed in the clinical setting, which were modeled in CD4+ T cell-depleted programmed death-1 receptor (PD-1) knockout B*57 : 01-Tg (B*57 : 01-Tg/PD-1-/-) mice. Here, we aimed to examine whether thymus and activation-regulated chemokine (TARC)/CCL17 level can be used as a biomarker for AHS. Serum TARC levels increased in HLA-B*57 : 01-transgenic mice following oral administration of ABC; this increase was associated with the severity of skin toxicity. In ABC-fed CD4+ T cell-depleted B*57 : 01-Tg/PD-1-/- mice, TARC was detected in the epidermal keratinocytes of the ear. Skin toxicity was characterized by the infiltration of CD8+ T cells partially expressing C-C chemokine receptor type 4, which is the primary receptor for TARC. In vivo TARC neutralization effectively alleviated the symptoms of ear skin redness and blood vessel dilatation. Moreover, TARC neutralization suppressed the infiltration of CD8+ T cells to the ear skin but did not affect the ABC-induced adaptive immune response. Therefore, TARC was involved in ABC-induced skin toxicity and contributed to the recruitment of CD8+ T cells to skin. This evidence suggests that serum TARC level may be a functional biomarker for AHS.

Weak complex formation of adverse drug reaction-associated HLAB57, B58, and B15 molecules.

The combination of certain human leukocyte antigen (HLA) polymorphisms with administration of certain drugs shows a strong correlation with developing drug hypersensitivity. Examples of typical combinations are HLA-B*57:01 with abacavir and HLA-B*15:02 with carbamazepine. However, despite belonging to the same serotype, HLA-B*57:03 and HLA-B*15:01 are not associated with drug hypersensitivity. Recent studies have shown that several HLA polymorphisms are associated with multiple drugs rather than a single drug, all resulting in drug hypersensitivity. In this study, we compared the molecular structures and intracellular localization of HLA-B*57:01, HLA-B*58:01, and HLA-B*15:02, which pose risks for developing drug hypersensitivity, as well as HLA-B*57:03 and HLA-B*15:01 that do not present such risks. We found that HLA molecules posing risks have a low affinity for the subunit ß2-microglobulin; notably, the weak hydrogen bond formed via Gln96 of the HLA molecule contributes to this behavior. We also clarified that these HLA molecules are easily accumulated in the endoplasmic reticulum, exhibiting a low expression on the cell surface. Considering that these hypersensitivity risk-associated HLA molecules form complexes with ß2-microglobulin and peptides in the endoplasmic reticulum, we assumed that their low complex formation ability in the endoplasmic reticulum facilitates the interaction with multiple drugs.

Regulation of the immune tolerance system determines the susceptibility to HLA-mediated abacavir-induced skin toxicity.

Idiosyncratic drug toxicity (IDT) associated with specific human leukocyte antigen (HLA) allotype is a rare and unpredictable life-threatening adverse drug reaction for which prospective mechanistic studies in humans are difficult. Here, we show the importance of immune tolerance for IDT onset and determine whether it is susceptible to a common IDT, HLA-B*57:01-mediated abacavir (ABC)-induced hypersensitivity (AHS), using CD4+ T cell-depleted programmed death-1 receptor (PD-1)-deficient HLA-B*57:01 transgenic mice (B*57:01-Tg/PD-1-/-). Although AHS is not observed in B*57:01-Tg mice, ABC treatment increases the proportion of cytokine- and cytolytic granule-secreting effector memory CD8+ T cells in CD4+ T cell-depleted B*57:01-Tg/PD-1-/- mice, thereby inducing skin toxicity with CD8+ T cell infiltration, mimicking AHS. Our results demonstrate that individual differences in the immune tolerance system, including PD-1highCD8+ T cells and regulatory CD4+ T cells, may affect the susceptibility of humans to HLA-mediated IDT in humans.

Increased penetration of diphenhydramine in brain via proton-coupled organic cation antiporter in rats with lipopolysaccharide-induced inflammation.

Uptake transporters in brain microvascular endothelial cells (BMECs) are involved in the penetration of basic (cationic) drugs such as diphenhydramine (DPHM) into the brain. Lipopolysaccharide (LPS)-induced inflammation alters the expression levels and activities of uptake transporters, which change the penetration of DPHM into the brain. A brain microdialysis study showed that the unbound brain-to-plasma partition coefficient (K p,uu,brain) for DPHM in LPS rats was approximately two times higher than that in control rats. The transcellular transport of DPHM to BMECs was increased when BMECs were cultured with serum from LPS rats. Compared with control rats or BMECs, the brain uptake of DPHM in LPS rats was increased and the intracellular accumulation of DPHM was increased under a high intracellular pH in BMECs from LPS rats, respectively. Treatment of BMECs with transporter inhibitors or inflammatory cytokines had little impact on the intracellular accumulation of DPHM in BMECs. This study suggests that LPS-induced inflammation promotes unidentified proton-coupled organic cation (H+/OC) antiporters that improve the penetration of DPHM into rat brain via the blood-brain barrier.

Increased brain penetration of diphenhydramine and memantine in rats with adjuvant-induced arthritis.

Brain penetration of cationic drugs is an important determinant of their efficacy and side effects. However, the effects of alterations in the activity of uptake transporters in the brain under inflammatory conditions on the brain penetration of cationic drugs are not fully understood. The aim of this study was to examine changes in brain penetration of cationic drugs, including diphenhydramine (DPHM), memantine (MMT), and cimetidine (CMD), and changes in the expression of uptake transporters such as organic cation transporter (Oct) in brain microvascular endothelial cells (BMECs) under inflammatory conditions. To clarify the effects of inflammation on the brain penetration of DPHM, MMT, and CMD, we performed brain microdialysis studies in a rat model of adjuvant-induced arthritis (AA). Further, differences in transporter mRNA expression levels between BMECs from control and AA rats were evaluated. Brain microdialysis showed that the unbound brain-to-plasma partition coefficient (Kp,uu,brain) for DPHM and MMT was significantly lower in AA rats compared with control rats. OCT mRNA levels were increased and proton-coupled organic cation (H+/OC) antiporter mRNA levels were decreased in AA rats compared with control rats. Taken together, our findings suggest that inflammation decreases the brain penetration of H+/OC antiporter substrates such as DPHM and MMT.

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice.

In receptor-type transcription factors-mediated cytochrome P450 (P450) induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels and metabolic activity of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A; PKN3 -/-] were determined after treatment with activators for receptor-type transcription factors. mRNA and protein levels and metabolic activity of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB) but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene compared with WT mice. We examined the CAR-dependent pathway regulated by PKN after PB treatment because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with Src kinase inhibitor 1 (SKI-1) or U0126 (a mitogen-activated protein kinase inhibitor) were evaluated. Treatment of hepatocytes from D mice with the combination of PB with U0126 but not SKI-1 significantly increased the mRNA levels of Cyp2b10 compared with those from the corresponding WT mice. These findings suggest that PKN may have inhibitory effects on the Src-receptor for activated C kinase 1 (RACK1) pathway in the CAR-mediated induction of Cyp2b10 in mice livers. SIGNIFICANCE STATEMENT: This is the first report of involvement of PKN in the transcriptional regulation of P450s. The elucidation of mechanisms responsible for induction of P450s could help optimize the pharmacotherapy and improve drug development. We examined whether the mRNA and protein levels and activities of P450s were altered in double-mutant mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations. PKN1/3 negatively regulates CAR-mediated induction of Cyp2b10 through phosphorylation of a signaling molecule in the Src-RACK1 pathway.

Changes in transporters and metabolizing enzymes in the livers of rats with bile duct ligation.

PURPOSE: Bile duct ligation (BDL) in experimental animals is widely used as an animal model of liver cholestasis and fibrosis. The transcriptional process and plasma membrane localization of transporters are regulated by nuclear receptors and scaffold proteins, respectively. However, the detailed changes of these factors in the livers of BDL rats remain unclear. To clarify the effects of BDL on the levels of transporters and metabolizing enzymes, nuclear receptors, and scaffold proteins, we investigated changes in mRNA and protein levels of livers from BDL rats. METHODS: Membrane proteins and microsomes were prepared from rats with BDL. The mRNA levels of transporters and nuclear receptors in livers of control and BDL rats were examined by real-time reverse transcription polymerase chain reaction. The protein levels of transporters, metabolizing enzymes and scaffold proteins in membrane proteins and microsomes were determined by liquid chromatography-tandem mass spectrometry-based targeted proteomics. RESULTS: Mdr1a mRNA was significantly decreased at 1 and 2 weeks of BDL. The mRNA levels of MRP2 were significantly decreased. The mRNA levels of nuclear receptors were significantly decreased in livers of 1-week BDL rats. The protein levels of P-gp were significantly increased by BDL. Regarding scaffold proteins, the protein levels of ezrin, moesin and EBP50 were significantly decreased at 2 weeks of BDL. The protein levels of radixin were significantly increased at 1 week of BDL. In 1-week BDL rats, the protein levels of metabolizing enzymes such as CYP and UGT were significantly decreased. CONCLUSIONS: This study reports the comprehensive changes of transporters, metabolizing enzymes, nuclear receptors, and ezrin/radixin/moesin proteins in the livers of BDL rats. The expression levels of nuclear receptors and radixin that regulate the transcription and localization of CYP and/or transporters were decreased by BDL.

Profiling of hepatic metabolizing enzymes and nuclear receptors in rats with adjuvant arthritis by targeted proteomics.

Inflammatory conditions alter the expression and activity of factors influencing pharmacokinetics, such as metabolizing enzymes. The study examined alterations of hepatic protein levels of cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and nuclear receptors in rats with adjuvant-induced arthritis (AA rats), an inflammatory animal model, by liquid chromatography-tandem mass spectrometry-based targeted proteomics. The protein levels of CYP1A1, CYP1A2, CYP2A1, CYP2A3, CYP2C6, CYP2C12, CYP2D3, CYP2E1, CYP3A9, UGT1A1 and UGT1A2/3 in liver microsomes of AA rats were significantly lower than those in control rats. The protein levels of constitutive androstane receptor (CAR) and retinoid X receptor α (RXRα) in the cytoplasm and nucleus were also significantly decreased, to approximately 60% of the control levels. The decreased protein levels of CYP1A2, CYP2C6, CYP2D3, CYP2E1 and UGT1A1 were potentially associated with downregulation of CAR or RXRα expression in the nucleus.