RESUMEN
Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and bacteraemia and is capable of remarkable phenotypic plasticity, responding rapidly to environmental change. Pneumococcus is a nasopharyngeal commensal, but is responsible for severe, acute infections following dissemination within-host. Pneumococcus is adept at utilising host resources, but the airways are compartmentalised and those resources are not evenly distributed. Challenges and opportunities in metabolite acquisition within different airway niches may contribute to the commensal-pathogen switch when pneumococcus moves from nasopharynx into lungs. We used NMR to characterise the metabolic landscape of the mouse airways, in health and during infection. Using paired nasopharynx and lung samples from naïve animals, we identified fundamental differences in metabolite bioavailability between airway niches. Pneumococcal pneumonia was associated with rapid and dramatic shifts in the lung metabolic environment, whilst nasopharyngeal carriage led to only modest change in upper airway metabolite profiles. NMR spectra derived from the nasopharynx of mice infected with closely-related pneumococcal strains that differ in their colonisation potential could be distinguished from one another using multivariate dimensionality reduction methods. The resulting models highlighted that increased branched-chain amino acid (BCAA) bioavailability in nasopharynx is a feature of infection with the high colonisation potential strain. Subsequent analysis revealed increased expression of BCAA transport genes and increased intracellular concentrations of BCAA in that same strain. Movement from upper to lower airway environments is associated with shifting challenges in metabolic resource allocation for pneumococci. Efficient biosynthesis, liberation or acquisition of BCAA is a feature of adaptation to nasopharyngeal colonisation.
Asunto(s)
Nariz , Infecciones Neumocócicas , Animales , Ratones , Metabolómica , Streptococcus pneumoniae , Aminoácidos de Cadena RamificadaAsunto(s)
Antibacterianos/efectos adversos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Haptenos/efectos adversos , Amoxicilina/efectos adversos , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/patología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Ácido Clavulánico/efectos adversos , Células Clonales , Floxacilina/efectos adversos , Expresión Génica , Antígenos HLA/genética , Antígenos HLA/inmunología , Humanos , Activación de Linfocitos/efectos de los fármacosRESUMEN
ß-Lactam antibiotics provide the cornerstone of treatment for respiratory exacerbations in patients with cystic fibrosis. Unfortunately, approximately 20% of patients develop multiple nonimmediate allergic reactions that restrict therapeutic options. The purpose of this study was to explore the chemical and immunological basis of multiple ß-lactam allergy through the analysis of human serum albumin (HSA) covalent binding profiles and T-cell responses against 3 commonly prescribed drugs; piperacillin, meropenem, and aztreonam. The chemical structures of the drug haptens were defined by mass spectrometry. Peripheral blood mononuclear cells (PBMC) were isolated from 4 patients with multiple allergic reactions and cultured with piperacillin, meropenem, and aztreonam. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ /IL-13 ELIspot. T-cell clones were generated from drug-stimulated T-cell lines and characterized in terms of phenotype, function, and cross-reactivity. Piperacillin, meropenem, and aztreonam formed complex and structurally distinct haptenic structures with lysine residues on HSA. Each drug modified Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent manner. PBMC proliferative responses and cytokine release were detected with cells from the allergic patients, but not tolerant controls, following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell clones isolated from the allergic patients were found to proliferate and release cytokines following stimulation with piperacillin, meropenem, or aztreonam. Cross-reactivity with the different drugs was not observed. In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific T-cell responses are readily detectable in allergic patients with cystic fibrosis, which indicates that multiple ß-lactam allergies are instigated through priming of naïve T-cells against the different drug antigens. Characterization of complex haptenic structures on distinct HSA lysine residues provides a chemical basis for the drug-specific T-cell response.
Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Hipersensibilidad a las Drogas/inmunología , Haptenos/química , Hipersensibilidad/inmunología , Albúmina Sérica/química , Linfocitos T/inmunología , Inhibidores de beta-Lactamasas/inmunología , beta-Lactamas/inmunología , Aztreonam/química , Aztreonam/inmunología , Fibrosis Quística/complicaciones , Fibrosis Quística/inmunología , Hipersensibilidad a las Drogas/complicaciones , Haptenos/inmunología , Humanos , Hipersensibilidad/complicaciones , Meropenem , Estructura Molecular , Piperacilina/química , Piperacilina/inmunología , Tienamicinas/química , Tienamicinas/inmunología , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/uso terapéutico , beta-Lactamas/química , beta-Lactamas/uso terapéuticoRESUMEN
UNLABELLED: The role of the adaptive immune system in adverse drug reactions that target the liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of human leukocyte antigen (HLA)-B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, we characterize flucloxacillin-responsive CD4+ and CD8+ T cells from patients with liver injury and show that naive CD45RA+CD8+ T cells from volunteers expressing HLA-B*57:01 are activated with flucloxacillin when dendritic cells present the drug antigen. T-cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL 25, and secreted interferon-gamma (IFN-γ), T helper (Th)2 cytokines, perforin, granzyme B, and FasL following drug stimulation. Flucloxacillin bound covalently to selective lysine residues on albumin in a time-dependent manner and the level of binding correlated directly with the stimulation of clones. Activation of CD8+ clones with flucloxacillin was processing-dependent and restricted by HLA-B*57:01 and the closely related HLA-B*58:01. Clones displayed additional reactivity against ß-lactam antibiotics including oxacillin, cloxacillin, and dicloxacillin, but not abacavir or nitroso sulfamethoxazole. CONCLUSION: This work defines the immune basis for flucloxacillin-induced liver injury and links the genetic association to the iatrogenic disease.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Floxacilina/efectos adversos , Antígenos HLA-B/fisiología , Activación de Linfocitos/inmunología , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Células Clonales/inmunología , Femenino , Floxacilina/metabolismo , Antígenos HLA-B/inmunología , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/efectos de los fármacos , Lisina/metabolismo , Masculino , Persona de Mediana Edad , Receptores CCR/biosíntesis , Receptores CCR4/biosíntesis , Albúmina Sérica/metabolismoRESUMEN
ß-Lactam antibiotics provide the cornerstone of treatment and reduce the rate of decline in lung function in patients with cystic fibrosis, but their use is limited by a high frequency of delayed-type allergic reactions. The objective of this study was to use cloned T-cells expressing a single T-cell receptor from five piperacillin-hypersensitive patients to characterize both the cellular pathophysiology of the reaction and antigen specificity to define the mechanism of activation of T-cells by piperacillin. More than 400 piperacillin-responsive CD4+, CD4+CD8+, or CD8+ T-cell clones were generated from lymphocyte transformation test and ELIspot-positive patients. The T-cell response (proliferation, T helper 2 cytokine secretion, and cytotoxicity) to piperacillin was concentration-dependent and highly specific. Enzyme-linked immunosorbent assay, gel electrophoresis, and mass spectrometry revealed that piperacillin bound exclusively to albumin in T-cell culture. Irreversible piperacillin binding at Lys 190, 195, 199, 432, and 541 on albumin and the stimulation of T-cells depended on incubation time. A synthetic piperacillin albumin conjugate stimulated T-cell receptors via a major histocompatibility complex- and processing-dependent pathway. Flucloxacillin competes for the same Lys residues on albumin as piperacillin, but the resulting conjugate does not stimulate T-cells, indicating that binding of the ß-lactam hapten in peptide conjugates confers structural specificity on the activation of the T-cell receptors expressed on drug-specific clones. Collectively, these data describe the cellular processes that underlie the structural specificity of piperacillin antigen binding in hypersensitive patients with cystic fibrosis.
Asunto(s)
Antibacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Fibrosis Quística/inmunología , Hipersensibilidad a las Drogas/inmunología , Piperacilina/inmunología , Adolescente , Adulto , Células Presentadoras de Antígenos/inmunología , Quimiocina CCL4/metabolismo , Células Clonales , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Femenino , Humanos , Interleucinas/metabolismo , Activación de Linfocitos , Masculino , Espectrometría de Masas , Receptores de Antígenos de Linfocitos T/metabolismo , Adulto Joven , beta-Lactamas/inmunologíaRESUMEN
A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.
Asunto(s)
Antígenos/sangre , Antígenos/fisiología , Fibrosis Quística/inmunología , Piperacilina/sangre , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Antígenos/biosíntesis , Línea Celular , Proliferación Celular/efectos de los fármacos , Cromatografía Liquida/métodos , Células Clonales , Fibrosis Quística/sangre , Hipersensibilidad a las Drogas/sangre , Hipersensibilidad a las Drogas/diagnóstico , Hipersensibilidad a las Drogas/inmunología , Femenino , Haptenos/biosíntesis , Haptenos/sangre , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/sangre , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/sangre , Piperacilina/farmacología , Unión Proteica/inmunología , Albúmina Sérica/biosíntesis , Albúmina Sérica/metabolismo , Albúmina Sérica/fisiología , Pruebas Cutáneas/métodos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
BACKGROUND: Exposure of patients with cystic fibrosis to sulfonamides is associated with a high incidence of hypersensitivity reactions. OBJECTIVE: To compare mechanisms of antigen presentation and characterize the phenotype and function of T cells from sulfamethoxazole-hypersensitive patients with and without cystic fibrosis. METHODS: T cells were cloned from 6 patients and characterized in terms of phenotype and function. Antigen specificity and mechanisms of antigen presentation to specific clones were then explored. Antigen-presenting cell metabolism of sulfamethoxazole was quantified by ELISA. The involvement of metabolism in antigen presentation was evaluated by using enzyme inhibitors. RESULTS: Enzyme inhibitable sulfamethoxazole-derived protein adducts were detected in antigen-presenting cells from patients with and without cystic fibrosis. A significantly higher quantity of adducts were detected with cells from patients with cystic fibrosis. Over 500 CD4(+) or CD8(+) T-cell clones were generated and shown to proliferate and kill target cells. Three patterns of MHC-restricted reactivity (sulfamethoxazole-responsive, sulfamethoxazole metabolite-responsive, and cross-reactive) were observed with clones from patients without cystic fibrosis. From patients with cystic fibrosis, sulfamethoxazole metabolite-responsive and cross-reactive, but not sulfamethoxazole-responsive, clones were observed. The response of the cross-reactive clones to sulfamethoxazole was dependent on adduct formation and was blocked by glutathione and enzyme inhibitors. Antigen-stimulated clones from patients with cystic fibrosis secreted higher levels of IFN-γ, IL-6, and IL-10, but lower levels of IL-17. CONCLUSION: Sulfamethoxazole metabolism and protein adduct formation is critical for the stimulation of T cells from patients with cystic fibrosis. T cells from patients with cystic fibrosis secrete high levels of IFN-γ, IL-6, and IL-10.