ABSTRACT
Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4(+) CD25(+-) forkhead box protein 3 (FoxP3)(+) and CD4(+) CD25(+) CD127(-) regulatory T (T(reg) ) cells when compared to NR-TB. Increased numbers of T(reg) cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-ß was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4(+) /interferon (IFN)-γ(+) T cells and enhanced CD4(+) CD25(+) FoxP3(+) , CD4(+) CD25(+) CD127(-) and CD4(+) CD25(+) IL-10(+) T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4(+) CD25(+) FoxP3(+) frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4(+) CD25(+) T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for T(reg) /T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.
Subject(s)
Immune Tolerance , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Tuberculosis, Multidrug-Resistant/immunology , Tuberculosis, Pulmonary/immunology , Adult , Aged , Antigens, Bacterial/immunology , Antigens, CD/metabolism , Bacterial Proteins/immunology , Cells, Cultured , Cytokines/immunology , Female , Forkhead Transcription Factors/metabolism , Humans , Immunophenotyping , Isoniazid/therapeutic use , Male , Middle Aged , Rifampin/therapeutic use , T-Lymphocyte Subsets/drug effects , T-Lymphocytes, Regulatory/drug effects , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/microbiology , Young AdultABSTRACT
BACKGROUND: Leprosy is characterized by a disease spectrum having two polar clinical forms dependent on the presence or not of cell-mediated immunity. In the tuberculoid forms, granuloma-activated macrophages kill Mycobacterium leprae in conjunction with a Th1 response while, in multibacillary (MB) lesions, M. leprae nonactivated macrophages infiltrate the nerves and internal organs together with a Th2 response. The functional properties and activation pathways of macrophages isolated from patients with MB leprosy remain only partially understood. OBJECTIVES: To establish an ex vivo methodology capable of evaluating the activation pathways, grade and fate of cultured macrophages isolated from MB lesions. METHODS: Skin biopsies from patients with borderline tuberculoid, bordeline lepromatous and lepromatous leprosy (LL) were characterized by immunohistochemistry and transcriptional analysis. To isolate inflammatory cells, a portion of the samples was submitted to enzymatic digestion. These same cells, maintained in culture for a minimum 7-day period, were characterized morphologically and via flow cytometry at different culture time points. Cytokine [interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10] mRNA levels were quantified by real-time polymerase chain reaction and protein secretion in the culture supernatants was measured by enzyme-linked immunosorbent assay and the nitric oxide levels by Griess reagent. RESULTS: RNA expression in tuberculoid and MB lesions showed the profile expected of characteristic Th1 and Th2 responses, respectively. The inflammatory cells in all biopsies were successfully isolated. Although the number of cells varied between biopsies, it was highest in LL biopsies. The frequency of isolated CD14+ and CD3+ cells measured by flow cytometry correlated with the percentages of macrophages and lymphocytes in the lesions. Throughout the culture period, CD68+ macrophages showed morphological changes. A progressive increase in cell number and reduction of infected cells were perceptible in the cultures. In contrast to the biopsies, TNF-alpha, IFN-gamma and IL-10 expression in the tuberculoid and MB leprosy cells in 24-h culture and the cytokine levels in the supernatants did not differ significantly. During the culture period, cytokine expression in the MB cells progressively declined, whereas, from days 1 to 7, nitrite levels progressively increased. After day 40, the remaining macrophages were able to ingest fluorescein isothiocyanate-labelled M. leprae. These data need to be confirmed. CONCLUSIONS: This study confirmed the feasibility of obtaining ex vivo macrophages from leprosy lesions and keeping them in long-term culture. This procedure may open new pathways to studying the interaction between M. leprae and human macrophages, which might, in turn, lead to the development of therapeutic tools capable of overcoming the specific anergy found in patients with MB leprosy.
