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Background. Multidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D-3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis. Methods. A clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, beta-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression). Results. VitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and I,C3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA-treated macrophages. Me combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid. Conclusions. Immunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis., Funding Agencies|Swedish Heart and Lung FoundationSwedish Heart-Lung Foundation [2019-0299, 2019-0302, 2017-0358, 2015-0236]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2019-01744, 201904720, 2016-01496, 2016-02043]; Foundation to Prevent Antibiotic Resistance (Resist); Lars Hierta Memorial Foundation; Karolinska Institutet FoundationsKarolinska Institutet; Karolinska InstitutetKarolinska Institutet

The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10 nM range, but no effect on bacterial growth or survival in the absence of host cells at 20 mu M. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling. (C) 2019 The Authors. Published by Elsevier Inc.

Background Intracellular delivery of antimicrobial agents by nanoparticles, such as mesoporous silica particles (MSPs), offers an interesting strategy to treat intracellular infections. In tuberculosis (TB), Mycobacterium tuberculosis avoids components of the immune system by residing primarily inside alveolar macrophages, which are the desired target for TB therapy. Methods and findings We have previously identified a peptide, called NZX, capable of inhibiting both clinical and multi-drug resistant strains of M. tuberculosis at therapeutic concentrations. In this study we analysed the potential of MSPs containing NZX for the treatment of tuberculosis. The MSPs released functional NZX gradually into simulated lung fluid and the peptide filled MSPs were easily taken up by primary macrophages. In an intracellular infection model, the peptide containing particles showed increased mycobacterial killing compared to free peptide. The therapeutic potential of peptide containing MSPs was investigated in a murine infection model, showing that MSPs preserved the effect to eliminate M. tuberculosis in vivo. Conclusions In this study we found that loading the antimicrobial peptide NZX into MSPs increased the inhibition of intracellular mycobacteria in primary macrophages and preserved the ability to eliminate M. tuberculosis in vivo in a murine model. Our studies provide evidence for the feasibility of using MSPs for treatment of tuberculosis., Funding Agencies|European Unions Seventh Framework Programme (FP7/2007-2013) [604182]; DFG [SFB 1279]; UK Medical Research Council; Swedish Heart-Lung Foundation [20150733]; Alfred Osterlunds Foundation; Royal Physiographic Society of Lund

Ethnopharmacological relevance: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need for continuous development of new and efficient methods to determine the susceptibility of isolates of Mycobacterium tuberculosis in the search for novel antimycobacterial agents. Natural products constitute an important source of new drugs, and design and implementation of antimycobacterial susceptibility testing methods are necessary to evaluate the different extracts and compounds. In this study we have explored the antimycobacterial properties of 50 ethanolic extracts from different parts of 46 selected medicinal plants traditionally used in Sudan to treat infectious diseases. Materials and methods: Plants were harvested and ethanolic extracts were prepared. For selected extracts, fractionation with hydrophilic and hydrophobic solvents was undertaken. A luminometry-based assay was used for determination of mycobacterial growth in broth cultures and inside primary human macrophages in the presence or absence of plant extracts and fractions of extracts. Cytotoxicity was also assessed for active fractions of plant extracts. Results: Of the tested extracts, three exhibited a significant inhibitory effect on an avirulent strain of Mycobacterium tubercluosis (H37Ra) at the initial screening doses (125 and 6.25 mu g/ml). These were bark and leaf extracts of Khaya senegalensis and the leaf extract of Rosmarinus officinalis L. Further fractions of these plant extracts were prepared with n-hexane, chloroform, ethyl acetate, n-butanol, ethanol and water, and the activity of these extracts was retained in hydrophobic fractions. Cytotoxicity assays revealed that the chloroform fraction of Khaya senegalensis bark was non-toxic to human monocyte-derived macrophages and other cell types at the concentrations used and hence, further analysis, including assessment of IC50 and intracellular activity was done with this fraction. Conclusion: These results e, Funding Agencies|Ekhaga Foundation [2011-33]; Swedish Insitute

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