RESUMEN
The role of CD4(+) T-cell interleukin-4 (IL-4) receptor alpha (IL-4Ralpha) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4(+) T-cell IL-4Ralpha knockout (KO) mice with the parasitic nematode Nippostrongylus brasiliensis, which induces strong host TH2 responses. Although N. brasiliensis expulsion was not affected in CD4(+) T-cell IL-4Ralpha KO mice, the associated lung pathology was reduced. Infected CD4(+) T-cell IL-4Ralpha KO mice showed abrogation of airway mucus production. Furthermore, CD4(+) T-cell IL-4Ralpha KO mouse lungs contained reduced numbers of lymphocytes and eosinophils. Restimulation of pulmonary region-associated T-cell populations showed that TH2 cytokine responses were disrupted. Secretion of IL-4, but not secretion of IL-13 or IL-5, from mediastinal lymph node CD4(+) T cells was reduced in infected CD4(+) T-cell IL-4Ralpha KO mice. Restimulation of tissue-derived CD4(+) T cells resulted in equivalent levels of IL-4 and IL-13 on day 7 postinfection (p.i.) in control and CD4(+) T-cell IL-4Ralpha KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4(+) T-cell IL-4Ralpha KO mice. Restimulation with N. brasiliensis antigen of total lung cell populations and populations with CD4(+) T cells depleted showed that CD4(+) T cells were a key TH2 cytokine source. These data demonstrated that CD4(+) T-cell IL-4 responsiveness facilitates eosinophil and lymphocyte recruitment, lymphocyte localization, and TH2 cytokine production in the allergic pathology associated with N. brasiliensis infections.
Asunto(s)
Interleucina-4/inmunología , Pulmón/patología , Infecciones por Strongylida/inmunología , Células Th2/inmunología , Animales , Quimiotaxis de Leucocito/inmunología , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Interleucina-4/deficiencia , Interleucina-4/genética , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Noqueados , Nippostrongylus/inmunología , Infecciones por Strongylida/microbiología , Infecciones por Strongylida/patologíaRESUMEN
Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed alpha/beta structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.