Inhalation of Respirable Crystalline Rifapentine Particles Induces Pulmonary Inflammation

Rifapentine is an anti-tuberculosis (anti-TB) drug with a prolonged half-life, but oral delivery results in low concentrations in the lungs because of its high binding (98%) to plasma proteins. We have shown that inhalation of crystalline rifapentine overcomes the limitations of oral delivery by significantly enhancing and prolonging the drug concentration in the lungs. The delivery of crystalline particles to the lungs may promote inflammation. This <i>in vivo</i> study characterizes the inflammatory response caused by pulmonary deposition of the rifapentine particles. The rifapentine powder was delivered to BALB/c mice by intratracheal insufflation at a dose of 20 mg/kg. The inflammatory response in the lungs and bronchoalveolar lavage (BAL) was examined at 12 h, 24 h, and 7 days post-treatment by flow cytometry and histopathology. At 12 and 24 h post-treatment, there was a significant influx of neutrophils into the lungs, and this returned to normal by day 7. A significant recruitment of macrophages occurred in the BAL at 24 h. Consistent with these findings, histopathological analysis demonstrated pulmonary vascular congestion and significant macrophage recruitment at 12 and 24 h post-treatment. In conclusion, the pulmonary delivery of crystalline rifapentine caused a transient neutrophil-associated inflammatory response in the lungs that resolved over 7 days. This observation may limit pulmonary delivery of rifapentine to once a week at a dose of 20 mg/kg or less. The effectiveness of weekly dosing with inhalable rifapentine will be assessed in murine <i>Mycobacterium tuberculosis</i> infection

Total Synthesis of Teixobactin

The first total synthesis of the cyclic depsipeptide natural product teixobactin is described. Synthesis was achieved by solid-phase peptide synthesis, incorporating the unusual l-<i>allo</i>-enduracididine as a suitably protected synthetic cassette and employing a key on-resin esterification and solution-phase macrolactamization. The synthetic natural product was shown to possess potent antibacterial activity against a range of Gram-positive pathogenic bacteria, including a virulent strain of <i>Mycobacterium tuberculosis</i> and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)

Nontoxic Metal–Cyclam Complexes, a New Class of Compounds with Potency against Drug-Resistant <i>Mycobacterium tuberculosis</i>

Tuberculosis (TB) accounted for 1.5 million deaths in 2014, and new classes of anti-TB drugs are required. We report a class of functionalized 1,8-disubstituted cyclam derivatives that display low micromolar activity against pathogenic mycobacteria. These compounds inhibit intracellular growth of <i>Mycobacterium tuberculosis</i>, are nontoxic to human cell lines, and are active against multidrug-resistant <i>M. tuberculosis</i> strains, indicating a distinct mode of action. These compounds warrant further appraisal as novel agents to control TB in humans

IDO-1 is dispensable for the control of M. tuberculosis infection in vivo.

<p>WT and IDO-1^−/− mice were infected with <i>M. tuberculosis</i> (100 CFU). IDO-1 expression in the lungs of uninfected WT mice (A), and WT (B) and IDO-1^−/− (C) mice at 28 days post infection. Lungs from uninfected IDO-1^−/− mice showed no IDO-1-positive cells (data not shown). IDO-1-expression in lungs of WT mice was primarily associated with inflammatory lesions within large monocytic cells (B), while IDO-1^−/− mice show no specific staining (C). One representative section from 1 of 5 mice per group. Bacterial loads (D) in infected lungs were determined at the time points indicated. Data represent the means +/− SD of 5 mice per group from one of two independent experiments. Lung inflammation (E) as percentage of the lung area with inflammatory cell infiltration from WT and IDO-1^−/− mice after subtracting the background cellularity in normal lung. To assess survival (F), infected mice were monitored daily and euthanized when showing signs of ill health. Data represent the time to euthanasia of 6 mice per group.</p

Normal cellular influx and the generation of antigen-specific T cells in the absence of IDO-1 in vivo.

<p>WT and IDO-1^−/− mice were infected with <i>M. tuberculosis</i> (100 CFU). Single cell suspensions were prepared from infected lungs and analyzed by flow cytometry for (A) CD4+, (B) CD8+ cells, and the percentages of these cells with an activated CD44hi/CD62lo phenotype ((C) CD4+, (D) CD8+). (E) Numbers of IFN-γ producing cells determined by ELISpot for lung cells cultured at 1×10⁵ cells/well with <i>M. tuberculosis</i> CFP overnight. Data represent the mean +/− SD of 5 mice from one representative of two experiments.</p

Mycobacteria and IFN-γ activate tryptophan-kynurenine metabolism in macrophages.

<p>(A) Human bronco-alveolar lavage cells were infected with <i>M. tuberculosis</i> (MOI 3) and stimulated with IFN-γ (100 and 500 U/ml) for 48 h. The concentration of quinolinic acid in cell culture supernatants was detected by electron-capture negative-ion gas chromatography-mass spectrometry. Data points are means +/− SD of triplicate wells representative of two independently performed experiments. Unpaired t test was used to compare raw data of three independent wells. (***P = 0.0003, **P<0.01, all other comparisons showed no significant difference) (B) Quinolinic and (C) picolinic acid concentrations in cell culture supernatants of human monocyte-derived macrophages stimulated overnight with IFN-γ (100 U/ml) before infection for 24, 48, and 72 h with <i>M. bovi</i>s BCG (MOI 5). Data points represent measurements obtained for cells from two individuals.</p

Inhibition of mycobacterial growth in vitro by picolinic acid.

<p>(A) <i>M. tuberculosis</i> was grown in 7H9 liquid medium in the presence of picolinic acid (100 - 1000 µM). Optical density of cultures was measured. Data are the mean +/− SD of four independent wells per condition and are representative of 2–4 independent experiments. <i>M. tuberculosis</i> CFU were determined upon exposure to picolinic acid (0.1 and 1 mM; HEPES and MES as buffer controls) in 7H9 liquid medium at pH 6.6 (B) and 5.5 (C). Data represent means +/− SD of triplicate wells. Similar pH-dependent effects of picolinic acid on <i>M. tuberculosis</i> survival were observed in three independent experiments.</p

<i>M. tuberculosis</i> infection increases IDO-1 expression in human and murine macrophages.

<p> (A) Microarray results of BAL cells (>90% macrophages) infected with <i>M. tuberculosis</i> (MOI 3–5) for 24 h compared to uninfected cells. Increased mRNA expression levels for Indoleamine 2,3-dioxygenase (IDO-1), Kynurenine 3-monooxygenase (KYN-OX), and L-Kynurenine hydrolase (KYN-HYD) are depicted as fold induction upon <i>M. tuberculosis</i> infection compared to uninfected control cells. Data are means +/− standard error of comparisons of microarray data obtained from four different individuals. (B) Murine bone marrow-derived macrophages were left untreated (1), infected with <i>M. tuberculosis</i> (MOI 5) (2) and stimulated with IFN-γ (100 U/ml) (3). Co-stimulation with both <i>M. tuberculosis</i> and IFN-γ was either performed simultaneously (4) or cells were pre-treated with IFN-γ for 24 h and then infected with <i>M. tuberculosis</i> for 24 h (5) before RNA isolation. IDO-1 mRNA expression and beta-2-microglobulin (control) were detected at 4, 24, and 48 h by RT-PCR. (6) H₂O. Data are representative of two independent experiments.</p

IDO-1 regulates CD4 T cell responses to mycobacterial antigen in vitro.

<p>Bone marrow-derived DCs were cultured for 4 days in vitro before stimulation for 2 h with Ag85 peptide P25. DCs were overlaid with CFSE labelled CD4⁺ T cells from P25-specific T cell receptor transgenic mice. Proliferation of T cells was determined by flow cytometry. DCs from WT and IDO-1^−/− mice were compared in their ability to induce proliferation of P25-specific T cells after 2 days (A, D) and 5 days (B, E). WT DCs were treated with 1 Methyl-DL-tryptophan before stimulation with P25 peptide. P25-specific T cell proliferation was determined after 5 days (C, F). (A-C) Fluorescence intensities of representative FACS plots are depicted. Grey- IDO-1^−/−/1-Methyl-DL-tryptophan, Black – WT/untreated control. The percentages of cells per division were calculated (D-F). Data represent the mean +/− SD of triplicate cultures from one of three representative experiments. The differences between groups were analysed by analysis of variance, *P<0.001.</p

Gene specific primers used in this study.

<p>Gene specific primers used in this study.</p