RESUMEN
BACKGROUND: Flucloxacillin (FLX)-induced liver injury is immune-mediated and highly associated to HLA-B∗57:01 expression. Host factors leading to drug-induced liver injury are not yet well understood. OBJECTIVE: Characterize in vivo immune mechanisms determining the development of CD8+ T cells reactive to FLX in animals expressing the risk human leukocyte antigen (HLA) allotype. METHODS: HLA-B∗57:01 transgenic mice (Tg) or Tg strains with H2-KbDb knockout (Tg/KO) or H2-KbDb/PD-1 double knockout (Tg/DKO) were treated with drug and/or anti-CD4 antibody. Drug-induced liver injury was evaluated on the basis of liver enzyme and histologic changes at day 10 of treatment. FLX-reactive CD8+ T cells were characterized in vitro by release of effector molecules on drug restimulation, gene expression, and flow cytometry analysis, and functionality tested for hepatic cytotoxicity. RESULTS: CD8+ T-cell responses to FLX in Tg were dependent on both HLA and mouse major histocompatibility complex I presentation and in vivo priming. Eliminating H2-KbDb in Tg/KO to allow exclusive presentation of FLX by HLA resulted in a less robust drug-specific CD8+T-cell response unless CD4+ cells, including regulatory T cells, were depleted. Treatment of Tg/KO with anti-CD4 antibody and FLX led to subclinical liver inflammation associated with an increase in PD1+CD8+ T cells in the lymphoid organs and liver. Impaired PD-1 expression in Tg/DKO led to liver histopathologic and transcriptional alterations but without hepatic enzyme elevations. Moreover, effector lymphocytes accumulated in the liver and showed FLX-dependent hepatic cytotoxicity in vitro when tolerogenic liver cells were depleted. CONCLUSIONS: In our in vivo models, FLX primes CD8+ T cells to recognize drug presented by HLA-B∗57:01 and murine major histocompatibility complex I. HLA-B∗57:01-dependent CD8+ T-cell reaction to FLX is limited by the presence of CD4+ cells, presumably regulatory T cells, and PD-1 expression. Tolerogenic hepatic cells limit clinical disease through PD-L1 or additional unexplored mechanisms.
Asunto(s)
Linfocitos T CD8-positivos , Enfermedad Hepática Inducida por Sustancias y Drogas , Ratones , Humanos , Animales , Floxacilina/efectos adversos , Floxacilina/metabolismo , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Antígenos de Histocompatibilidad Clase I , Ratones Transgénicos , Antígenos HLA/genética , Modelos Animales de Enfermedad , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismoRESUMEN
Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a ß-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.
Asunto(s)
Floxacilina/inmunología , Antígenos HLA-B/inmunología , Haptenos/inmunología , Péptidos/inmunología , Animales , Línea Celular , Antígenos HLA-B/genética , Humanos , Ratones , Ratones Transgénicos , Péptidos/genéticaRESUMEN
Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.
Asunto(s)
Didesoxinucleósidos/efectos adversos , Tolerancia a Medicamentos/genética , Tolerancia a Medicamentos/inmunología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/inmunología , Antígenos HLA-B/genética , Animales , Fármacos Anti-VIH/efectos adversos , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Hipersensibilidad a las Drogas/genética , Hipersensibilidad a las Drogas/inmunología , Femenino , Humanos , Técnicas In Vitro , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de la Transcriptasa Inversa/efectos adversosRESUMEN
Microbes induce innate immune responses in hosts. It is critical to know how different microbes control adaptive responses through innate pathways. The impact of gram-positive bacteria on the innate and adaptive responses is unclear. Herein we report that Staphylococcus aureus induces IL-10, Th17-inducing cytokines IL-6 and IL-23, chemokines, and regulates dendritic cell markers. S. aureus inhibits T-cell IL-2 responses through modulation of HLA-DR, CD86 and PD-L1. IFN-gamma, Src kinase inhibitors, or TLR2 antibodies prevented the down-modulation of HLA-DR by S. aureus. Our data demonstrate that innate TLR signaling induces multi-dimensional inhibition of adaptive immune responses, which may contribute to the lack of protective immunity to bacteria or microbe tolerance. IL-10 and PD-L1 antagonists may boost immunity to vaccines for S. aureus and other microbes.
Asunto(s)
Inmunidad Adaptativa , Antígeno B7-H1/metabolismo , Interleucina-10/metabolismo , Staphylococcus aureus/inmunología , Receptor Toll-Like 2/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Antígeno B7-H1/inmunología , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Humanos , Mediadores de Inflamación/inmunología , Interleucina-10/genética , Macrófagos/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Linfocitos T/inmunología , Receptor Toll-Like 2/genética , Transcripción Genética , Familia-src Quinasas/metabolismoRESUMEN
Ag presentation is a key step in the initiation of adaptive immune responses that depends on the expression of MHC Ags and costimulatory molecules. Immune-enhancing CpG and non-CPG oligodeoxynucleotides (ODNs) stimulate Ag presentation by stimulating the expression of these molecules and by promoting dendritic cell maturation. In this report, we identify immunoregulatory orthophosphorothioate non-CpG molecules, referred to as regulatory ODNs (rODNs), by their ability to inhibit allogeneic monocyte-stimulated T cell responses and down-regulate HLA-DR in human primary monocytes. The rODNs promoted the survival of macrophages and were able to activate IL-8 secretion through a chloroquine-resistant pathway. Messenger RNAs for HLA-DR alpha and beta and the MHC CIITA were reduced by rODNs but not by stimulatory CpG ODN2006 and non-CpG ODN2006a. CIITA transcription in monocytes was controlled primarily by promoter III and not by promoter I or IV. rODNs blocked promoter III-directed transcription of CIITA in these cells. Under conditions that induced dendritic cell differentiation, rODNs also reduced HLA-DR expression. The activity of rODNs is phosphorothioate chemistry and G stretch dependent but TLR9 independent. G tetrads were detected by circular dichroism in active rODNs and associated with high m.w. multimers on nondenaturing gels. Heat treatment of rODNs disrupted G tetrads, the high m.w. aggregates, and the HLA-DR inhibitory activity of the ODNs. The inhibition of immune responses by regulatory oligodeoxynucleotides may be useful for the treatment of immune-mediated disorders including autoimmune diseases and graft rejection.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Regulación hacia Abajo/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Oligodesoxirribonucleótidos/farmacología , Regiones Promotoras Genéticas/inmunología , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Proliferación Celular , Células Cultivadas , Inhibidores de Crecimiento/fisiología , Antígenos HLA-DR/biosíntesis , Antígenos HLA-DR/metabolismo , Cadenas alfa de HLA-DR , Humanos , Isoantígenos/farmacología , Proteínas Nucleares/biosíntesis , Regiones Promotoras Genéticas/genética , Linfocitos T/citología , Linfocitos T/inmunología , Transactivadores/biosíntesis , Transcripción Genética/inmunologíaRESUMEN
Chemokine receptor CXCR4 (CD184) may play a role in cancer metastasis and is known to form homodimers. However, it is not clear how transmembrane regions (TM) of CXCR4 and receptor homotypic interactions affect the function of CXCR4 in living cells. Using confocal microscopy and flow cytometric analysis, we showed that high levels of CXCR4 are present in the cytoplasm, accompanied by lower expression on the cell surface in CXCR4 transfectants, tumor cells, and normal peripheral blood lymphocytes. CXCR4 homodimers were detected in tumor cells, both on the cell surface membrane and in the cytoplasm using fluorescence resonance energy transfer and photobleaching fluorescence resonance energy transfer to measure energy transfer between CXCR4-CFP and CXCR4-YFP constructs. Disruption of lipid rafts by depletion of cholesterol with methyl-beta-cyclodextrin reduced the interaction between CXCR4 molecules and inhibited malignant cell migration to CXCL12/SDF-1alpha. A synthetic peptide of TM4 of CXCR4 reduced energy transfer between molecules of CXCR4, inhibited CXCL12-induced actin polymerization, and blocked chemotaxis of malignant cells. TM4 also inhibited migration of normal monocytes toward CXCL12. Reduction of CXCR4 energy transfer by the TM4 peptide and methyl-beta-cyclodextrin indicates that interactions between CXCR4s may play important roles in cell migration and suggests that cell surface and intracellular receptor dimers are appropriate targets for control of tumor cell spread. Targeting chemokine receptor oligomerization and signal transduction for the treatment of cancer, HIV-1 infections, and other CXCR4 mediated inflammatory conditions warrants further investigation.
Asunto(s)
Quimiotaxis , Péptidos/farmacología , Receptores CXCR4/metabolismo , Actinas/metabolismo , Proteínas Bacterianas/genética , Línea Celular , Línea Celular Tumoral , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiotaxis/efectos de los fármacos , Colesterol/metabolismo , Citoplasma/metabolismo , Dimerización , Transferencia de Energía , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Humanos , Ligandos , Proteínas Luminiscentes/genética , Linfocitos/metabolismo , Microdominios de Membrana/metabolismo , Microscopía Confocal , Péptidos/química , Receptores CXCR4/química , Receptores CXCR4/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Chemokines attract leukocytes bearing the relevant chemokine receptors and regulate innate immune responses. CpG oligodeoxynucleotides (ODN) and GM-CSF are potent vaccine adjuvants and in combination induce enhanced Th1 responses by mechanisms yet to be determined. We have examined combinations of CpG- or non-CpG-ODN and GM-CSF for effects on the production of chemokines and the differentiation of monocytes to dendritic cells. High levels of the Th1-attracting, HIV-1-inhibitory chemokines, CCL3/MIP-1alpha and CCL4/MIP-1beta, were induced in human primary monocytes when CpG- or non-CpG-ODN was combined with GM-CSF, but not with IL-4 or IFN-gamma. The synergistic induction of beta-chemokines by non-CpG-ODN was phosphorothioate (PS) chemistry dependent and inhibited by blocking endosome maturation/acidification and ERK1/2 activation. Chemokine and TLR9 mRNAs were induced by PS-ODN. Cells treated with non-CpG PS-ODN and GM-CSF expressed dendritic cell marker CD83 and high levels of HLA-DR and costimulatory molecules, and were CD14(-) or CD14(dim), consistent with monocyte differentiation into a dendritic cell phenotype. The induction of CD83 and beta-chemokines was tyrosine phosphorylation dependent. Secreted CCL3 and CCL4 were detected as a heterodimer. Our results indicate the CpG-independent synergy between PS-ODN and GM-CSF mediated through chemokine and dendritic cell induction. In addition, our observations suggest that PS-ODN plus GM-CSF may be useful as potent ex vivo dendritic cell differentiation/maturation agents for dendritic cell therapy and as vaccine adjuvants for tumor and infectious microorganisms, including HIV-1.
Asunto(s)
Quimiocinas CC/biosíntesis , Islas de CpG/inmunología , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Monocitos/inmunología , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/síntesis química , Tionucleótidos/química , Antígenos CD/biosíntesis , Antígeno B7-2 , Antígenos CD40/biosíntesis , Diferenciación Celular/inmunología , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Proteínas de Unión al ADN/biosíntesis , Células Dendríticas/citología , Células Dendríticas/metabolismo , Regulación hacia Abajo/inmunología , Sinergismo Farmacológico , Endosomas/inmunología , Endosomas/metabolismo , Antígenos HLA-DR/biosíntesis , Humanos , Inmunoglobulinas/biosíntesis , Inmunofenotipificación , Receptores de Lipopolisacáridos/metabolismo , Proteínas Inflamatorias de Macrófagos/biosíntesis , Proteínas Inflamatorias de Macrófagos/genética , Glicoproteínas de Membrana/biosíntesis , Monocitos/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptor Toll-Like 9 , Antígeno CD83RESUMEN
Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1alpha. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine-chemokine receptor interactions and HIV-1 coreceptor functions.
Asunto(s)
Antígenos CD4/fisiología , VIH-1/fisiología , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Animales , ADN Viral/análisis , Regulación hacia Abajo , Endocitosis , Ratones , Células 3T3 NIH , TransfecciónRESUMEN
Activated lymphocytes synthesize and secrete substantial amounts of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha/CCL3 and MIP-1 beta/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1 beta secreted by activated human peripheral blood lymphocytes (MIP-1 beta(3-69)) lacks the two NH(2)-terminal amino acids of the full-length protein. This truncated form of MIP-1 beta has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1 beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1 beta(3-69) showed that the truncated protein not only shares the ability of intact MIP-1 beta to induce Ca(2+) signaling through CCR5, but unlike the full-length protein, it also triggers a Ca(2+) response via CCR1 and CCR2b. These results demonstrate that NH(2)-terminally truncated MIP-1 beta functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.