Correlation between Central Memory T Cell Expression and Proinflammatory Cytokine Production with Clinical Presentation of Multibacillary Leprosy Relapse

<div><p>Background</p><p>Despite the efficacy of multidrug therapy, surviving <i>Mycobacterium leprae</i> causes relapse in some leprosy patients, and these patients present signs and symptoms of disease after healing. This study focused on the cellular immune response in relapsed multibacillary patients but also included non-relapsed multibacillary cured individuals, newly diagnosed and untreated multibacillary patients, paucibacillary patients just before the beginning of treatment, and voluntary healthy individuals for comparative analysis.</p><p>Methodology/Principal Findings</p><p>Inhibition of CD86 expression in the blood-derived monocytes and dendritic cells of relapsed multibacillary patients, either <i>ex vivo</i> or after <i>M</i>. <i>leprae</i> antigen stimulation was observed by flow cytometry. In addition, no significant changes in Interferon-gamma (IFN-γ) expression were observed in 5-day culture supernatants of relapsed patients in response to <i>M</i>. <i>leprae</i>, neither before nor after treatment, as measured by ELISA. However, these patients demonstrated a significant increase in central memory CD4+ and CD8+ <i>M</i>. <i>leprae</i>-specific T cells, as assessed by multiparametric flow cytometry. The increase in frequency of central memory T cells in relapsed patients strongly correlated with the bacillary index and the number of skin lesions observed in these subjects. Moreover, cytokine multiplex analysis demonstrated significant antigen-specific production of Interlukin-1beta (IL-1b), IL-6, and Tumour Necrosis Factor (TNF) in the relapsed group with extremely low IL-10 production, which resulted in a high TNF/IL-10 ratio.</p><p>Conclusions/Significance</p><p>Inhibition of CD86 expression may function to reduce effector T cell responses against the <i>M</i>. <i>leprae</i> antigen. Furthermore, the predominance of central memory T cells in association with the high TNF/IL-10 ratio and no observed IFN-γ production may be related to the pathogenesis of relapse in multibacillary leprosy. Therefore, our findings may be a direct result of the clinical presentation, including a number of skin lesions and bacterial load, of relapsed patients. To our knowledge, this is the first study correlating immune response parameters with the clinical presentation of relapsed multibacillary patients.</p></div

Significant inhibition of CD86 expression in monocytes and DC from MB relapsed leprosy patients.

<p>PBMC cultures stimulated with <i>M</i>. <i>leprae</i> (20 μg/mL) and LPS (1 μg/mL) for 24 h were analyzed by flow cytometry (FACSAria, BD). After gating the monocytes and DC region, based on forward and side scatter and the negative region defined with isotype control antibodies for each cell population, CD86 expression was obtained both in monocytes (CD3-/CD83-/CD14+; A) and DC (CD3-/CD14-/CD83+; B). On the left, the bars of both graphics correspond to antigen stimulation, while those on the right correspond to LPS stimulation, in accordance with the assessed groups. The results are expressed as percentage variation, calculated according to the following formula: [CD86 expression in ML-stimulated cultures (%) / CD86 expression in unstimulated cultures (%) × 100] −100; C). A representative example of the flow cytometric determination of CD86 expression in unstimulated (UNS), <i>M</i>. <i>leprae</i> stimulated (ML), and LPS stimulated monocytes, and DC from a relapsed patient (upper dot plots) and a healthy volunteer (lower dot plots). The numbers inside the dot plots represent the percentage of CD86 positive cells in monocytes (CD83-; lower right panels) and DC (CD83+; upper right panels).</p

Baseline patterns of newly diagnosed and untreated patients, cured leprosy patients, and healthy volunteers.

<p>Baseline patterns of newly diagnosed and untreated patients, cured leprosy patients, and healthy volunteers.</p

Increase in the frequency of CD4+ and CD8+ TCM lymphocytes in relapsed patients is related to BI and to the number of skin lesions.

<p>CD4+ and CD8+ TCM cells (CD69+/CD45RO+/CD62L+high; A and D) in response to in vitro 24-h PBMC stimulation with M. leprae (20 μg/mL) by multiparametric flow cytometry (FACSAria, BD). The results are expressed as percentage of positive cells, and the statistical differences are shown (***p< 0.001; UNS: unstimulated). The horizontal bars represent the median of each group. In each analysis, isotype controls were used to distinguish between positive and negative cells. SEB (1μg/mL) was positive in all tests (data not shown). The Kruskal-Wallis test was used for comparison of stimulated cells with unstimulated cells, and the Mann–Whitney test to group comparisons. Correlation analysis between CD4+ and CD8+ TCM and BI (B and E) and the number of lesions (C and F). Significant positivity is demonstrated (r = correlation coefficient; p= significance level; Pearson test).</p

The cytokine profiles in response to <i>M</i>. <i>leprae</i>, TNF, and IL-10 are differentially induced in PBMC during relapse.

<p>Detection of IL-1β (A), IL-6 (B), IFN-γ (C), TNF (D) and IL-10 (E) in supernatant cultures was performed by multiplex assay. The results are expressed in pg/mL and the statistical differences are shown (*<i>p<</i>0.05, **<i>p<</i> 0.005 and ***<i>p<</i> 0.001; the Kruskal-Wallis test was used for comparison of stimulated cells with unstimulated cells and the Mann–Whitney test was used to group comparisons). UNS: unstimulated and ML: <i>M</i>. <i>leprae</i> stimulated cultures (20 μg/mL). MB: untreated multibacillary; HC: healthy controls. The horizontal bars represent the median of each group. SEB (1 μg/mL) was positive in all tests (data not shown). A high <i>M</i>. <i>leprae</i>-specific TNF/IL-10 ratio between relapsed patients and other groups, especially newly diagnosed and untreated MB patients was observed (F).</p

Characteristics of multibacillary patients at the FLD and at relapse.

<p>Characteristics of multibacillary patients at the FLD and at relapse.</p

Effector molecule levels increase in response to <i>M</i>. <i>leprae</i> after index case treatment and BCG vaccination of household contacts of MB leprosy patients (HCMB).

<p>Levels of effector molecules in supernatants of 5-day cultures stimulated with ML (A) or a pool of HLA Class II-restricted ML-specific synthetic peptides (B) were evaluated by multiplex assays in supernatants of 5-day cultures of peripheral blood leukocytes of HCMB before (T0) and after (T1) treatment of their index cases and BCG vaccination. Box plots show median, interquartile range, sample minimum, and maximum levels. Dots represent individual donors. n = 16.</p

Effector molecule levels increase in response to <i>M</i>. <i>leprae</i> after index case treatment and BCG vaccination of household contacts of MB leprosy patients (HCMB).

<p>Levels of effector molecules in supernatants of 5-day cultures stimulated with ML (A) or a pool of HLA Class II-restricted ML-specific synthetic peptides (B) were evaluated by multiplex assays in supernatants of 5-day cultures of peripheral blood leukocytes of HCMB before (T0) and after (T1) treatment of their index cases and BCG vaccination. Box plots show median, interquartile range, sample minimum, and maximum levels. Dots represent individual donors. n = 16.</p

Study design.

<p>HCMB were enrolled during a period of 0–3 months after the beginning of their index case treatment. The first blood collection was performed prior to BCG vaccination according to the presence/absence of a BCG scar (T0). After an interval of 6 to 26 months from chemotherapy onset of the index case, the same individuals were asked to provide new peripheral blood samples (T1).</p

Epidemiological data of MB leprosy patient household contacts.

<p>Epidemiological data of MB leprosy patient household contacts.</p