Gr1^int and Gr1^hi cells sorted from <i>M. tuberculosis</i> infected NOS-/- mice activate Th17 cells and induce proliferation of CD4⁺ T cells.

<p>Specific populations of Gr1^int or Gr1^hi cells were sorted from infected NOS-/- mice and placed into <i>in </i><i>vitro</i> cultures with increasing numbers of either stimulated naïve or infected splenocytes. After 2 days of incubation T cells were evaluated for activation and memory profiles (A-D). Panel A -D shows lower ratios of Gr1^high (A and C) or Gr1^int (B and D) induce increased the percentage of activation of CD4⁺CD69⁺IL-17⁺ and memory CD4⁺CD44⁺IL-17⁺, when co-incubated with naïve (A and B) or <i>M. tuberculosis</i> infected splenocytes (C and D), respectively. Panel E and F shows Gr1 ^high and Gr1^int induced of proliferating CD4 T cells. However, as the splenocyte to Gr1^high or Gr1^int ratio increased the percentage of CD4+ proliferation was reduced. Results are expressed as the mean values of mean percentage of CD4⁺CD69⁺IL-17⁺, CD4⁺CD44⁺IL-17⁺ and proliferation. ANOVA and the Tukey post-test. *p<0.05, **p<0.01, ***p<0.001.</p

Higher numbers of CD11b⁺ cells in the lungs of NOS2-/- KO mice.

<p>Flow cytometry analysis was performed on single cell suspensions obtained from lungs of WT C57/BL6 or NOS2-/- mice. In these mice increased numbers of CD11b⁺ cells but not CD11b⁺CD11c⁺ cells could be observed as the infection progressed (A, B, C). In contrast, differences for CD11c⁺ and CD4⁺ cells only became evident at later time points (B, D). No significant differences were observed for CD8⁺ cells (E). Results are expressed as the mean values of log mean cell number (± SEM, n=5) in the lung. Bacterial burden (F) was enumerated at different time points after a low dose aerosol infection with <i>M. tuberculosis</i>. As observed, bacterial burden in both murine strains is similar at early time points after infection and diverges after 30 days of infection. Results are expressed as the mean Log 10 CFUs (±SEM, n=5) in the lungs. Student t-test, * p<0.05, ** p<0.01, *** p<0.001.</p

Significant numbers of Gr1^int CD11b⁺ cells were also found in RAG2-/- and C3HeB/FeJ mice.

<p>The influx of Gr1^intCD11b⁺ cells was evaluated in immunocompromised RAG-/- (A), and immunocompetent C3HeB/FeJ mice (C) and NOS-/- (F). Again, large numbers of Gr1^intCD11b⁺ cells were observed in these three mouse strains and significant differences were observed in the Gr1⁺ MFI (B, D, G). Similar to NOS2 -/- mice, arginase activity was also increased in C3HeB/FeJ undergoing lung necrosis (E). In contrast, C3H/HeOuJ did not have major arginase activity. Similar to NOS2 -/- mice, arginase activity was also increased in C3HeB/FeJ undergoing lung necrosis (E). Results are expressed as the mean values of mean fluorescence intensity (MFI) or arginase activity (± SEM, n=5) in the Lung. **Student t-test, p<0.001. </p

Gr1^int CD11b⁺ FSC^low cells predominate in the lungs of NOS2-/- mice.

<p>Further analysis of CD11b⁺FSC^low and CD11b⁺FSC^high cells was performed by staining with anti-Gr1 antibody. As determined by mean fluorescence intensity, CD11b⁺FSC^high had significantly higher Gr1 expression than CD11b⁺FSC^low (A). Starting at early time points NOS2-/- mice but not WT C57BL/6 mice have a significant number of cells giving an intermediate staining pattern for Gr-1 (B). At later time points, the influx of CD11b⁺FSC^high cells into the lungs of NOS2 -/- mice is also enhanced (C). Results are expressed as the Log mean cell number (± SEM, n=5) in the lung. Gr1^high CD11b⁺ FSC^high cells express markers compatible with a monocytic lineage. Both populations of Gr1 expressing cells were further analyzed by flow cytometry using the monocytic markers Ly6C, CD14 and F4/80 (D). As determined by the mean fluorescence intensity for these markers, a more precise phenotype for these two cellular populations would be Gr1^high CD11b⁺ FSC^high Ly6C^high CD14⁺ F4/80⁺ and Gr1^int CD11b⁺ FSC^low Ly6C^low CD14^low F4/80^low, compatible with a monocytic and granulocytic lineage, respectively. Results are expressed as the log mean cell number (± SEM, n=5) in the Lung. ***Student t test, * p<0.05, ** p<0.01, *** p<0.001.</p

Gr1^int and Gr1^hi cells sorted from <i>M. tuberculosis</i> infected NOS-/- mice expressed arginase I and IL-17.

<p>Specific populations of Gr1^int or Gr1^hi cells were sorted from infected NOS-/- mice in an attempt to further characterize Gr1^int and Gr1^hi cell function during tuberculosis using flow cytometry. Panel A, demonstrates pre-sorted Gr1+CD11b+ cells which were then further characterized. Gr1^hi and Gr1^int sorted cells (B, C) both demonstrated high expression of arginase I and IL-17. Panel D shows the isotype controls for arginase-1 and IL-17A. </p

Peripheral localization and distinct morphology of Gr1⁺ cells present in necrotic lungs.

<p>Anti-Gr1 immunohistochemistry was performed to determine the location of Gr1⁺ cells surrounding necrotic lesions in the NOS2 -/- mice. As observed in low magnification (4x), anti-Gr1 labeling was predominantly localized in the periphery or rim of these structures (red, arrows, A). At higher magnification (100x), two distinct cellular morphologies could be observed for cells staining with anti-Gr1. In the left panel, neutrophils with multi-lobed nucleus could readily be seen. However, particularly in close apposition to the fibrotic capsule, cells were seen that had abundant and vacuolated cytoplasms as well as a unilobed nucleus, consistent with the morphology of MDSCs. Arginase activity was biochemically detected in the lungs of NOS2 -/- mice but minimally WT controls (B). As disease progressed in NOS2 -/-, more arginase activity could be detected compared to wild-type mice (B).Results are expressed as the mean values of arginase activity (± SEM, n=5) in the Lung. **Student t-test, p<0.01 and ***Student t-test, p<0.001. Anti-arginase-1 immunohistochemistry was performed to determine its expression by inflammatory cells in the lungs of NOS2-/- mice. Similar to the anti-Gr1 staining, at low magnification (4x), robust arginase-1 expression was detected in cells located in the rim of necrotic granulomas (C, left panel). At higher magnification (100x), arginase-1 expression was limited to highly vacuolated cells containing abundant cytoplasm (C, right panel). Panel D shows co-localized staining of both Gr1+ and arginase-1 in NOS2 -/- mouse lung tissues after 45 days of infection. At higher magnification (100x), co-localized staining of Gr1+ and arginase-1 demonstrates that Gr1+ cells (D lower right panel, arrow, red) and arginase-1 staining (D, arrow, brown) are clearly co-localized on the same cell. </p

Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of <i>Mycobacterium tuberculosis</i> in Human Macrophages

<div><p></p><p>Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to <i>Mycobacterium tuberculosis</i> (<i>MTB</i>). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to <i>MTB</i>. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular <i>MTB</i>. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular <i>MTB</i> in human macrophages via induction of apoptosis and autophagy.</p></div

TNFα and IFNγ levels in MDM and AM infected with <i>MTB</i> H37Rv with or without NFκB inhibition.

<p>Primary human (<b>A</b>) MDM or (<b>B</b>) AM were infected with <i>MTB</i> H37Rv and after 1 hr, 24 hrs, 4 days, or 8 days of infection, supernatants were assayed for TNFα by ELISA and IFNγ by electrochemiluminescence. Data shown are estimated means with standard error bars from linear mixed model fits, based on seven independent experiments (MDM) or nine independent experiments (AM). *p<0.05 and **p<0.01 for cytokine expression in macrophages infected with <i>MTB</i> alone (closed circles) vs. <i>MTB</i>+BAY (closed squares). Control = (open circles) and BAY = (open squares).</p

Inhibition of NFκB activation induces autophagy in THP-1 cells.

<p>(<b>A</b>) Control THP-1 cells (0.1% DMSO) and THP-1 cells subjected to serum starvation, 5 µM BAY, <i>MTB</i> infection, or both <i>MTB</i>+BAY for 24 hrs, followed by immunoblotting of nuclear-free whole cell lysates for LC3 and β-actin. A representative immunoblot of three independent experiments is shown. (<b>B</b>) Human THP-1 cells were transduced with lentivirus-GFP-LC3 and differentiated into macrophages, followed by infection with <i>MTB</i> for 24 hrs in the absence or presence of 5 µM BAY. The cells were fixed and stained with DAPI to visualize the nuclei (blue) and the number of GFP-positive punctae were quantified. <i>Upper panel</i>, representative immunofluorescence images of three independent experiments; <i>lower panel</i>, average number of GFP-LC3 punctae per cell. The data shown represent the mean ± SEM of duplicate wells/condition from three independent experiments. (<b>C</b>) <i>MTB</i>-infected THP-1 cells treated with BAY were incubated with or without 3-MA, an inhibitor of the early phase of the autophagic pathway. After 48 hrs, the cells were lysed and nuclear-free whole cell lysates (20 µg per lane) were separated by SDS-PAGE and immunoblotted for LC3-I, LC3-II and β-actin. The bar graph represents the relative densities of the LC3-II bands normalized for their corresponding β-actin bands for two independent experiments. (<b>D</b>) THP-1 cells were infected with <i>MTB</i> H37Rv alone, <i>MTB</i>+5 µM BAY, or <i>MTB</i>+BAY+6 mM 3-MA for 4 days and cell-associated <i>MTB</i> was quantified. Data shown are mean ± SEM from two independent experiments performed in duplicates. *p<0.05, **p<0.01, ***p<0.001.</p

<i>MTB</i> H37Rv induces NFκB binding to its <i>cis</i>-regulatory element.

<p>(<b>A</b>) THP-1 cells were pre-treated with 0.1% (v/v) DMSO or 5 µM BAY for 1 hr, followed by infection with <i>MTB</i> H37Rv at the indicated times. An EMSA was performed with an oligonucleotide that corresponds to the consensus binding sequence for NFκB. Data shown are representative of three independent experiments. (<b>B</b>) Primary human MDM or (<b>C</b>) AM were pre-incubated with 0.1% DMSO or 5 µM BAY for 1 hr, then infected with <i>MTB</i> for 3 hrs, and followed by an EMSA to assay for NFκB binding. Data shown are representative of two independent experiments for MDM and AM. NS = non-specific band. SS = supershift band.</p