Journal: Cellular microbiology

Article Title: NAD hydrolysis by the tuberculosis necrotizing toxin induces lethal oxidative stress in macrophages

doi: 10.1111/cmi.13115

Figure Lengend Snippet: THP-1 macrophages were infected with Mtb strains at an MOI of 10:1 and treated with N-acetylcysteine (NAC, 100 μM) when indicated. ROS levels were measured with the fluorescent probe H2DCFDA at (A) 4, 24 and 48 h or (B) 48 h post-infection. (C) Cell viability of infected macrophages was measured 48 h after infection as the total ATP content with a luminescent ATP detection assay kit. (D) The Mtb intracellular growth in infected macrophages was measured at 4 h and 48 h post-infection and expressed as CFU per ml. (E) Wt Mtb H37Rv was grown in Middlebrook 7H9 medium with 0.5% glycerol, 0.02% Tyloxapol and 10% OADC and supplemented with N-acetyl-cysteine (NAC, 100 μM). The OD600 was measured at the indicated time points. Asterisks indicate significant differences (p-value<0.01, calculated using the Two-way ANOVA with Bonferroni’s correction) compared with the indicated conditions. Data are represented as mean ± SEM.

Article Snippet: Mtb H37Rv and its derivative strains were grown in Middlebrook 7H9 medium (Difco) supplemented with 0.5% glycerol, 0.02% Tyloxapol and 10% OADC (Remel) or in Middlebrook 7H10 plates supplemented with 0.5% glycerol and 10% OADC (Remel).

Techniques: Infection, Detection Assay

Journal: Immunology

Article Title: Myeloid HIF‐1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection

doi: 10.1111/imm.13131

Figure Lengend Snippet: Myeloid‐deficient hypoxia‐inducible factor‐1 alpha (mHIF‐1α−/−) mice are more susceptible to chronic Mycobacterium tuberculosis (Mtb) infection. B6 and mHIF‐1α−/− mice were challenged with different doses of the H37Rv Mtb strain via the aerosol route. (a) Survival of B6 and mHIF‐1α−/− mice challenged with 500 CFU of Mtb infection. Data represent 10 animals per group from two independent experiments each with five mice per group. (b) Lung bacterial burdens of mice challenged with Mtb (75 CFU) at 25 and 40 days post‐infection (dpi). (c) Mice challenged with Mtb via the 150 CFU in experiment 1, 75 CFU in experiment 2, and 500 CFU in experiment 3. At 60 and 120 dpi, bacterial burdens were assessed in the lungs. Data represent the mean CFU ± SD from four−nine mice per group. Individual plots from experiments 2 and 3 are from two independent assays each with four−five mice per group. Statistical significance was calculated by using unpaired Student’s t‐test with a Welch correction (*P < 0·05; ns, not significant).

Article Snippet: Aerosol infections with Mtb strains H37Rv (originally from the Trudeau Institute) or HN878 were carried out using the Glas‐Col airborne infection system, as previously described.

Techniques: Infection, Aerosol

Journal: Immunology

Article Title: Myeloid HIF‐1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection

doi: 10.1111/imm.13131

Figure Lengend Snippet: Hypoxia‐inducible factor 1‐alpha (HIF‐1α) activity in the myeloid compartment regulates pulmonary inflammation during chronic Mycobacterium tuberculosis (Mtb) infection. B6 and myeloid‐deficient (m)HIF‐1α−/− mice were challenged with Mtb (H37Rv) infection via the aerosol route (experiments 2 and 3). (a) Representative haematoxylin & eosin (H&E) lung sections at 60 and 120 days post‐infection (dpi). (b) Percentage of the infiltrated area in the lungs at 60 and 120 dpi for three experiments. Individual data points represent one lung section. Data represent the mean ± SD from six−10 mice per group. Individual plots from experiments 2 and 3 are from two independent assays each with four−five mice per group. Statistical significance was calculated by using unpaired Student’s t‐test with a Welch correction (*P < 0·05; **P < 0·01).

Article Snippet: Aerosol infections with Mtb strains H37Rv (originally from the Trudeau Institute) or HN878 were carried out using the Glas‐Col airborne infection system, as previously described.

Techniques: Activity Assay, Infection, Aerosol

Journal: Immunology

Article Title: Myeloid HIF‐1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection

doi: 10.1111/imm.13131

Figure Lengend Snippet: Expression of inducible nitric oxide synthase (iNOS) in lungs from wild‐type (WT) and myeloid‐deficient hypoxia‐inducible factor‐1 alpha (mHIF‐1α−/−) mice at 60 and 120 days post‐infection (dpi) with Mycobacterium tuberculosis (Mtb) H37Rv (experiments 2 and 3). (a) Representative images of the immunohistochemical analysis of iNOS in lung tissues of Mtb‐infected mice. (b) Optical density quantification of iNOS staining. Data represent the mean ± SD of eight−10 mice per group. Individual plots from experiments 2 and 3 are from two independent assays each with four−five mice per group. Statistical significance was calculated by using unpaired Student’s t‐test with a Welch correction (*P < 0·5; ns, not significant).

Article Snippet: Aerosol infections with Mtb strains H37Rv (originally from the Trudeau Institute) or HN878 were carried out using the Glas‐Col airborne infection system, as previously described.

Techniques: Expressing, Infection, Immunohistochemical staining, Staining

Journal: Immunology

Article Title: Myeloid HIF‐1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection

doi: 10.1111/imm.13131

Figure Lengend Snippet: Gene expression at 60 and 120 dpi (Mtb H37Rv) in the lungs of WT and mHIF‐1α −/− mice

Article Snippet: Aerosol infections with Mtb strains H37Rv (originally from the Trudeau Institute) or HN878 were carried out using the Glas‐Col airborne infection system, as previously described.

Techniques: Gene Expression, Significance Assay

Journal: Immunology

Article Title: Myeloid HIF‐1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection

doi: 10.1111/imm.13131

Figure Lengend Snippet: Analysis of different cell populations in the lungs of WT and mHIF‐1α −/− mice at 60 dpi (Mtb H37Rv)

Article Snippet: Aerosol infections with Mtb strains H37Rv (originally from the Trudeau Institute) or HN878 were carried out using the Glas‐Col airborne infection system, as previously described.

Techniques: Significance Assay

Journal: Immunology

Article Title: Myeloid HIF‐1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection

doi: 10.1111/imm.13131

Figure Lengend Snippet: Myeloid hypoxia‐inducible factor‐1 alpha (HIF‐1α) deficiency prompts enhanced CD4+ T‐cell responses in myeloid‐deficient (m)HIF‐1α−/− mice challenged with Mycobacterium tuberculosis (Mtb) H37Rv (experiments 2 and 3). (a) Lung CD4+ T‐cell numbers determined by flow cytometry at 60 days post‐infection (dpi). (b−d) Lung cells were restimulated with ESAT‐61‐20 to assess the polyfunctionality of CD4+ T‐cells. (b) Representative flow cytometry data of interferon‐gamma (IFN‐γ)‐, tumour necrosis factor‐alpha (TNF‐α)‐ and interleukin (IL)‐17‐expressing CD4+ T‐cells. (c) Frequencies and (d) numbers of IFN‐γ+ single producers (SP), IFN‐γ+TNF‐α+ double producers (DP), TNF‐α+ SP and IL‐17 SP CD4+ T‐cells. (e) IFN‐γ concentration in the supernatants of lung single cell suspensions at 60 dpi determined by ELISA. Data represent the mean ± SD of nine−10 mice per group. Individual plots from experiments 2 and 3 are from two independent assays each with four−five mice per group. Statistical significance was calculated by using unpaired Student’s t‐test with a Welch correction (*P < 0·05; **P < 0·01; ***P < 0·01; ****P < 0·0001; ns, not significant).

Article Snippet: Aerosol infections with Mtb strains H37Rv (originally from the Trudeau Institute) or HN878 were carried out using the Glas‐Col airborne infection system, as previously described.

Techniques: Flow Cytometry, Infection, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay