BCG vaccination induces an early Th1 cytokine profile in <i>M. ulcerans</i>-infected footpads.

<p>Mice were either non-immunized (•) or immunized with BCG (○) two months before challenge. All mice were infected in the footpad with 4 log₁₀ CFU of <i>M. ulcerans</i> 98–912. At different times post-infection, total RNA from the footpad was extracted and the presence of mRNA for IFN-ã (A), TNF (B), MIP2 (C) was assessed by real-time PCR. Data points represent the mean ± SEM (<i>n</i> = 5–8) for each time point. Statistical significance was calculated with Student's <i>t</i> test (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001).</p

Immunization increases iNOS positivity in the footpad of <i>M. ulcerans</i> challenged mice.

<p>Mice were either non-immunized (A and D) or immunized with BCG (B, E, and G) or with mycolactone-negative <i>M. ulcerans</i> 5114 (C, F, and H) two months before challenge. All mice were infected in the footpad with 2 log₁₀ CFU of <i>M. ulcerans</i> 98–912. The footpads were harvested at on day 60 (A–C), day 70 (D–F), or day 100–200 (G–H and J–K) and processed for histology. Cells were stained for the presence of iNOS (red) and nuclei were stained with DAPI (blue). In footpads of non-immunized mice, scarce or no iNOS positive cells were found in the tissue (A, D). The footpads of vaccinated mice presented foci of iNOS positive cells during the initial weeks of infection (B–C and E–F). With progression of infection, iNOS positivity became sparse. Images are representative of 4 footpads per group. Original magnification: 20×.</p

Vaccination with BCG induces an early CD4⁺ T cell response in the DLN of <i>M. ulcerans</i>-infected mice.

<p>Mice were either non-immunized (•) or immunized with BCG (○) two months before challenge. All mice were infected in the footpad with 4 log₁₀ CFU of <i>M. ulcerans</i> 98–912. At different time points post-infection, the total number of leukocytes in the popliteal lymph node of the infected footpad was counted (A) and the number of CD4⁺ (B), and CD8⁺ (C) cells was determined by flow cytometry. The data points represent the mean ± SEM (<i>n</i> = 5–8). Statistical significance was calculated with Student's <i>t</i> test (*<i>p</i><0.05; **<i>p</i><0.01).</p

Immunization with BCG or mycolactone-negative <i>M. ulcerans</i> delays the onset of footpad swelling after challenge with a low dose of virulent <i>M. ulcerans</i>.

<p>Mice were either non-immunized (•) or immunized with BCG (○) or with <i>M. ulcerans</i> 5114 (▵) two months before challenge. All mice were infected in the footpad with 2 log₁₀ CFU of <i>M. ulcerans</i> 98–912. Footpad swelling was monitored throughout the experimental infection (<i>n</i> = 10). Initial footpad swelling was considered at 2.60 mm, after which mice progressed to ulceration of the footpad. For ethical reasons, mice were sacrificed upon onset of footpad ulceration.</p

Vaccination with BCG induces an early IFN-γ response in the DLN of <i>M. ulcerans</i>-infected mice.

<p>Mice were either non-immunized (•) or immunized with BCG (○) two months before challenge. All mice were infected in the footpad with 4 log₁₀ CFU of <i>M. ulcerans</i> 98–912. (A) At different time points post-infection, the total number of CD3⁺ CD4⁺ IFN-γ⁺ in the DLN of the infected footpad was determined by flow cytometry. The data points represent the mean ± SEM (<i>n</i> = 5–8). (B) At different times post-infection, total RNA from the popliteal lymph node was extracted and the presence of mRNA for IFN-γ was assessed by real-time PCR. Data points represent the mean ± SEM (<i>n</i> = 5–8) for each time point. Statistical significance was calculated with Student's <i>t</i> test (*<i>p</i><0.05). Statistical significance was calculated with Student's <i>t</i> test (*<i>p</i><0.05; **<i>p</i><0.01).</p

Immunization induces a transient mononuclear inflammatory response in the footpad of <i>M. ulcerans</i> challenged mice.

<p>Mice were either non-immunized (A, D, and I) or immunized with BCG (B, E, G, and J) or with mycolactone-negative <i>M. ulcerans</i> 5114 (C, F, H, K) two months before challenge. All mice were infected in the footpad with 2 log₁₀ CFU of <i>M. ulcerans</i> 98–912. The footpads were harvested at on day 60 (A–C), day 70 (D–F and I), or day 150–200 (G–H and J–K) and processed for histology. Tissue sections were stained with HE (A–H) or ZN (I–K). (A, D, and I) Non-immunized mice showed a persistent mixed inflammatory infiltrate composed of mononuclear cells (arrowheads) and neutrophils (arrows). Necrotic areas harbored abundant extracellular bacilli (I) and cells with apoptotic morphology (triangles). (B, C, E, F, G, H, J, and K) The footpads of vaccinated mice presented a mononuclear inflammatory infiltrate (arrowheads) throughout the initial weeks of experimental infection, which was later replaced by a predominantly neutrophilic infiltrate (arrows) and/or apoptosis (triangles) and extracellular bacilli (J, K). Images are representative of 4 footpads per group. Original magnification: 10×. Original magnification of HE inserts: 100×. Original magnification of ZN: 100×.</p

Immunization with BCG or mycolactone-negative <i>M. ulcerans</i> 5114 delays the destruction of the DLN during infection with virulent <i>M. ulcerans</i>.

<p>Mice were either non-immunized (A, D, and I) or immunized with BCG (B, E, G, and J) or with mycolactone-negative <i>M. ulcerans</i> 5114 (C, F, H, K) two months before challenge. All mice were infected in the footpad with 2 log₁₀ CFU of <i>M. ulcerans</i> 98–912. The DLN were recovered at day 60 (A–C), day 70 (D–F and I), and days 100–200 (G–H and J–K) and processed for histology. Tissue sections were stained with HE (A–H) or ZN (I–K). (A, D, and I) In non-immunized mice infection resulted in extensive areas of necrosis and bacterial colonization by day 70 post-infection. (B, C, E, F, G, H, J, and K) Immunized mice showed no alterations in the structure of the DLN during these first 70 days. As the infectious process progressed in immunized mice, bacilli could be found throughout the tissue (arrowheads) along with signs of tissue destruction. Images are representative of 4 lymph nodes per group analyzed. Original magnification (A–H): 4×. Original magnification (I–K): 100×.</p

Ablation of myeloid Sirt2 transiently impacts the control of <i>M</i>. <i>tuberculosis</i>.

<p>(A) Lung and (B) liver <i>M</i>. <i>tuberculosis</i> burdens at days 30, 60 and 120 post-infection of Cre⁺Sirt2^fl/fl mice (white circles) or Cre^-Sirt2^fl/fl (black circles). Represented are 3 independent experiments. The initial infectious dose was Log₁₀1.942±0.106; Log₁₀2.00±0.030; and Log₁₀2.177±0.124, for three independent experiments performed. *, p < 0.05; **, p < 0.01; determined by unpaired t-test; (C) Microscopic inflammatory lung lesions of <i>M</i>. <i>tuberculosis</i>-infected mice stained with hematoxylin-eosin. (D) NOS2 (red) and nuclei (blue) immunofluorescence staining, 30 days post-infection.</p

Absence of Sirt2 in myeloid cells does not impact lung cellular responses to <i>M</i>. <i>tuberculosis</i>.

<p>(A) Myeloid cell populations in lung 30 days post-infection were characterized by flow cytometry. (B) Flow cytometry analysis of total CD4+ T cells and IFN-γ, IL-17 and TNF production by CD4+ T cells restimulated with PMA and ionomycin in the presence of brefeldin A. Graphs show the mean ± SEM value of one representative experiment of at least two independent ones (n = 5). The gating strategies and representative plots are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131904#pone.0131904.s001" target="_blank">S1 Fig</a>. Significance was determined by the Student’s <i>t</i>-test.</p

The expression of inflammatory mediators in infected lungs is not altered in the absence of myeloid Sirt2.

<p>RNA was extracted from the lung tissue after 30 days of infection and the expression of <i>Ifn</i>γ, <i>Il17</i>, <i>Tnf</i>, <i>Il6</i> and <i>Nos2</i> was analyzed by real-time PCR and normalized to the expression of ubiquitin. Data shows the mean ± SEM value (n = 5–6) and the significance was determined by the Student’s <i>t</i>-test. The data are representative of two independent experiments.</p