Production of IFNγ by lymphocytes stimulated with ESAT-6 (Panel A) and CFP-10 (Panel B) was tested in the ELISPOT technique.

<p>The groups of patients with inactive TB (n = 5), active TB (n = 6) and healthy vaccinated subjects (n = 13) were tested. Cell-mediated immunological responses were determined as the number of reactive cells per 10⁶ lymphocytes. The lower dotted line is the level of positivity suggested by the manufacturer, the upper dotted line is the level of positivity adopted at the HUSLAB diagnostic laboratory. The area between the two dotted lines represents the so-called grey-zone, an area of uncertainty for interpretation that was calculated based on assay imprecision (data not published).</p

ROC curves were constructed to compare ELISPOT results when the cells were stimulated with rMtb-HBHA (A), PPD (B), and peptide mixtures of ESAT-6 and CFP-10 (C–D).

<p>The curves were established for infected (Active and Inactive TB) and the healthy control group. The calculated AUC and the respective confidence intervals (in brackets) are shown for each tested antigen.</p

Characteristics of the individuals enrolled in the study.

<p>Characteristics of the individuals enrolled in the study.</p

The ability of rMtb-HBHA and PPD to induce the production of IFNγ was tested in the ELISPOT technique.

<p>The groups of patients with inactive TB (n = 5); active TB (n = 6), healthy young (n = 7) and middle-age (n = 8) vaccinated subjects and patients with isolation of so-called atypical mycobacteria (n = 4) and were enrolled. Comparison of the cell-mediated immunological responses to rMtb-HBHA was performed as the determination of the number of reactive cells per10⁶ lymphocytes (A) and as the measurement of IFNγ production activities expressed as arbitrary units (B). Cell-mediated immunological responses in healthy vaccinated individuals was studied by division by age into two groups (C). The correlation of rMtb-HBHA with the PPD ELISPOT reactivities expressed as the frequencies of reactive cells per10⁶ is presented in (D). Data are shown as individual reactivities; the horizontal bars represent arithmetic median values.</p

DataSheet_1_Mycobacterial acyl carrier protein suppresses TFEB activation and upregulates miR-155 to inhibit host defense.pdf

Mycobacterial acyl carrier protein (AcpM; Rv2244), a key protein involved in Mycobacterium tuberculosis (Mtb) mycolic acid production, has been shown to suppress host cell death during mycobacterial infection. This study reports that mycobacterial AcpM works as an effector to subvert host defense and promote bacterial growth by increasing microRNA (miRNA)-155-5p expression. In murine bone marrow-derived macrophages (BMDMs), AcpM protein prevented transcription factor EB (TFEB) from translocating to the nucleus in BMDMs, which likely inhibited transcriptional activation of several autophagy and lysosomal genes. Although AcpM did not suppress autophagic flux in BMDMs, AcpM reduced Mtb and LAMP1 co-localization indicating that AcpM inhibits phagolysosomal fusion during Mtb infection. Mechanistically, AcpM boosted the Akt-mTOR pathway in BMDMs by upregulating miRNA-155-5p, a SHIP1-targeting miRNA. When miRNA-155-5p expression was inhibited in BMDMs, AcpM-induced increased intracellular survival of Mtb was suppressed. In addition, AcpM overexpression significantly reduced mycobacterial clearance in C3HeB/FeJ mice infected with recombinant M. smegmatis strains. Collectively, our findings point to AcpM as a novel mycobacterial effector to regulate antimicrobial host defense and a potential new therapeutic target for Mtb infection.</p

Effect of eIF2α phosphorylation on intracellular survival of Mtb H37Rv.

<p>Raw264.7 cells were pretreated for 30 min with indicated concentrations of salubrinal and then infected with Mtb H37Rv (MOI = 1) for 3 h. Salubrinal remained for the rest of the infection. (A) The cells were incubated for 48 h and Western blot analysis was performed using antibodies directed against CHOP, p-eIF2α and β-actin. DMSO alone was used as the negative control. (B) Quantification of intracellular survival of Mtb H37Rv in Raw264.7 cells pretreated for 30 min with salubrinal as described above. The cells were collected at 48 h postinfection with Mtb H37Rv and bacteria number was determined by CFU counting. The statistical significance (*<i>P</i><0.05) of observed differences between salubrinal treated and untreated groups following infection with Mtb H37Rv were verified by two-tailed t-test. Data represent the mean±standard error of the mean (SEM) of values obtained in three independent experiments.</p

Mtb H37Rv infection induces ER stress sensor proteins in macrophage cells and CHOP is activated in BMDMs or A549 cells.

<p>Cells were infected with Mtb H37Rv (MOI = 1) for 3 h, and then incubated for 0–48 h. Immunoblot analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028531#s4" target="_blank">Materials and Methods</a>. The statistical significance (*<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001) of observed differences between Mtb H37Rv infected and uninfected groups were verified by two-tailed t-test. (A) Raw264.7 cells, (B) BMDMs or A549 cells. Representative data from three independent experiments are shown. STS: staurosporine.</p

The expression of ER stress markers in Raw264.7 cells infected with live or heat-killed Mtb H37Rv.

<p>Raw264.7 cells were infected with live or heat killed Mtb H37Rv (MOI = 1 to 10) for 3 h, and then incubated for 48 h in the presence or absence of L-NAME or NAC. (A, D, E) Immunoblot analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028531#s4" target="_blank">Materials and Methods</a>. Representative data from three independent experiments are shown. (B) Effect of Mtb H37Rv infection on nitric oxide (NO) production was assessed indirectly by Griess reaction. (C) Representative flow cytometry histograms of superoxide at 48 h after Mtb H37Rv infection. Superoxide detection was evaluated by dihydroethidium (DHE) staining using flow cytometry. Data are means ± SEM of two independent experiments performed in triplicate. CHOP expression analysis after treatment with N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide inhibitor (D) and N-acetyl-L-cysteine (NAC), a superoxide inhibitor (E). The statistical significance (*<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001) of observed differences between inhibitor treated and untreated groups following infection with Mtb H37Rv were verified by two-tailed t-test.</p

<i>Mycobacterium tuberculosis</i> Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential

<div><p>Macrophages constitute the first line of defense against <i>Mycobacterium tuberculosis</i> and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from <i>M</i>. <i>tuberculosis</i> culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-κB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against <i>M</i>. <i>tuberculosis</i> in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development.</p></div

<i>Ex vivo</i> Rv2882c stimulation induced Ag-specific memory T cell expansion in spleen and lung cells from Mtb-infected mice.

<p>The cells were prepared as described the Materials and Methods. (A) Gating strategy to assess intracellular cytokines and transcription factors. All samples were stained for surface molecules and gated based on forward scatter (FSC) and side scatter (SSC). The T cells were gated from the lymphocyte gate by FSC vs. SSC based on surface expression patterns of CD4⁺ T cells. Using the CD4⁺ T cell gate, cells specific staining for CD62L and CD44 are shown in stimulated spleen or lung cells. The BMDMs (1 × 10⁵/well) were treated with Rv2882c (10 μg/mL) or LPS (100 ng/mL) for 24 h. Next, media or CD4⁺ T cells (1 × 10⁶/well) from the lungs (B, D) or spleens (C, E) of Mtb-infected mice were added to each well and incubated for 3 days. (B and C) The numbers of naïve CD4⁺ T cells (CD4⁺CD44^-CD62L⁺) and effector/memory CD4⁺ T cells (CD4⁺CD44⁺CD62L^-) were determined by flow cytometry. (D and E) The levels of IFN-γ and IL-2 were determined by ELISA. The data shown are the mean values ± SD (n = 15); *<i>p</i> < 0.05, **<i>p</i> < 0.01 or ***<i>p</i> < 0.001 = a significant difference between Rv2882c-treated and untreated or Rv2882c-treated and Ag85B-treated cells, as determined by one-way ANOVA. Treatments with no significant effect are indicated by <i>n</i>.<i>s</i>.</p