Publications
42 results found
Asai M, Li Y, Newton S, et al., 2023, Galleria mellonella-intracellular bacteria pathogen infection models: the ins and outs, FEMS Microbiology Reviews, ISSN: 0168-6445
Gollnick H, Barber J, Wilkinson RJ, et al., 2023, IL-27 inhibits anti- Mycobacterium tuberculosis innate immune activity of primary human macrophages., Tuberculosis (Edinb), Vol: 139
Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen that primarily infects macrophages. Despite a robust anti-mycobacterial response, many times macrophages are unable to control M. tuberculosis. The purpose of this study was to investigate the mechanism by which the immunoregulatory cytokine IL-27 inhibits the anti-mycobacterial activity of primary human macrophages. We found concerted production of IL-27 and anti-mycobacterial cytokines by M. tuberculosis-infected macrophages in a toll-like receptor (TLR) dependent manner. Notably, IL-27 suppressed the production of anti-mycobacterial cytokines TNFα, IL-6, IL-1β, and IL-15 by M. tuberculosis-infected macrophages. IL-27 limits the anti-mycobacterial activity of macrophages by reducing Cyp27B, cathelicidin (LL-37), LC3B lipidation, and increasing IL-10 production. Furthermore, neutralizing both IL-27 and IL-10 increased the expression of proteins involved in LC3-associated phagocytosis (LAP) pathway for bacterial clearance, namely vacuolar-ATPase, NOX2, and RUN-domain containing protein RUBCN. These results implicate IL-27 is a prominent cytokine that impedes M. tuberculosis clearance.
Bachanová P, Cheyne A, Broderick C, et al., 2022, Comparative transcriptomic analysis of whole blood mycobacterial growth assays and tuberculosis patients’ blood RNA profiles, Scientific Reports, Vol: 12, Pages: 1-13, ISSN: 2045-2322
In vitro whole blood infection models are used for elucidating the immune response to Mycobacterium tuberculosis (Mtb). They exhibit commonalities but also differences, to the in vivo blood transcriptional response during natural human Mtb disease. Here, we present a description of concordant and discordant components of the immune response in blood, quantified through transcriptional profiling in an in vitro whole blood infection model compared to whole blood from patients with tuberculosis disease. We identified concordantly and discordantly expressed gene modules and performed in silico cell deconvolution. A high degree of concordance of gene expression between both adult and paediatric in vivo-in vitro tuberculosis infection was identified. Concordance in paediatric in vivo vs in vitro comparison is largely characterised by immune suppression, while in adults the comparison is marked by concordant immune activation, particularly that of inflammation, chemokine, and interferon signalling. Discordance between in vitro and in vivo increases over time and is driven by T-cell regulation and monocyte-related gene expression, likely due to apoptotic depletion of monocytes and increasing relative fraction of longer-lived cell types, such as T and B cells. Our approach facilitates a more informed use of the whole blood in vitro model, while also accounting for its limitations.
Asai M, Li Y, Spiropoulos J, et al., 2022, Galleria mellonella as an infection model for the virulent Mycobacterium tuberculosis H37Rv, Virulence, Vol: 13, Pages: 1543-1557, ISSN: 2150-5594
Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable and advantageous additional model to be used alongside existing models to advance tuberculosis research.
Asai M, Sheehan G, Li Y, et al., 2021, Innate immune responses of Galleria mellonella to Mycobacterium bovis BCG challenge identified using proteomic and molecular approaches, Frontiers in Cellular and Infection Microbiology, Vol: 11, ISSN: 2235-2988
The larvae of the insect Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes (gloverin, cecropin, IMPI, hemolin, and Hdd11), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.
Asai M, Li Y, Spiropoulos J, et al., 2020, A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella, Virulence, Vol: 11, Pages: 811-824, ISSN: 2150-5594
Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialised animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.
Singh Khara J, Mojsoska B, Mukherjee D, et al., 2020, Ultra-short antimicrobial peptoids show propensity for membrane activity against multi-drug resistant Mycobacterium tuberculosis, Frontiers in Microbiology, Vol: 11, Pages: 1-11, ISSN: 1664-302X
Tuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.
Asai M, Li Y, Singh Khara J, et al., 2019, Galleria mellonella: a novel infection model for screening potential anti-mycobacterial compounds against members of the Mycobacterium tuberculosis complex, Frontiers in Microbiology, Vol: 10, ISSN: 1664-302X
Drug screening models have a vital role in the development of novel antimycobacterial agents which are urgently needed to tackle drug-resistant tuberculosis (TB). We recently established the larvae of the insect Galleria mellonella (greater wax moth) as a novel infection model for the Mycobacterium tuberculosis complex. Here we demonstrate its use as a rapid and reproducible screen to evaluate antimycobacterial drug efficacy using larvae infected with bioluminescent Mycobacterium bovis BCG lux. Treatment improved larval survival outcome and, with the exception of pyrazinamide, was associated with a significant reduction in in vivo mycobacterial bioluminescence over a 96 hour period compared to the untreated controls. Isoniazid and rifampicin displayed the greatest in vivo efficacy and survival outcome. Thus G. mellonella, infected with bioluminescent mycobacteria, can rapidly determine in vivo drug efficacy, and has the potential to significantly reduce and/or replace the number of animals used in TB research.
Asai M, Li Y, Khara J, et al., 2019, Use of the invertebrate Galleria Mellonella as an infection model to study the Mycobacterium tuberculosis complex, Jove-Journal of Visualized Experiments, Vol: 148, ISSN: 1940-087X
Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world’s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging.Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.
Li Y, Spiropoulos J, Cooley J, et al., 2018, Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex, Virulence, Vol: 9, Pages: 1126-1137, ISSN: 2150-5594
Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the M. tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent, Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection
von Both U, Berk M, Agapow P-M, et al., 2018, Mycobacterium tuberculosis Exploits a Molecular Off Switch of the Immune System for Intracellular Survival, Scientific Reports, Vol: 8, ISSN: 2045-2322
Mycobacterium tuberculosis (M. tuberculosis) survives and multiplies inside human macrophages by subversion of immune mechanisms. Although these immune evasion strategies are well characterised functionally, the underlying molecular mechanisms are poorly understood. Here we show that during infection of human whole blood with M. tuberculosis, host gene transcriptional suppression, rather than activation, is the predominant response. Spatial, temporal and functional characterisation of repressed genes revealed their involvement in pathogen sensing and phagocytosis, degradation within the phagolysosome and antigen processing and presentation. To identify mechanisms underlying suppression of multiple immune genes we undertook epigenetic analyses. We identified significantly differentially expressed microRNAs with known targets in suppressed genes. In addition, after searching regions upstream of the start of transcription of suppressed genes for common sequence motifs, we discovered novel enriched composite sequence patterns, which corresponded to Alu repeat elements, transposable elements known to have wide ranging influences on gene expression. Our findings suggest that to survive within infected cells, mycobacteria exploit a complex immune “molecular off switch” controlled by both microRNAs and Alu regulatory elements.
Hemingway C, Berk M, Anderson ST, et al., 2017, Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function., PLoS ONE, Vol: 12, ISSN: 1932-6203
The WHO estimates around a million children contract tuberculosis (TB) annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM). Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB), and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB) and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68%) of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16) and PTB patients (r2 = 0.71 p = 2x10-16) when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11) and T-cell receptor signalling (p = 6.56E-07). Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003). Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies.
Reuschl AK, Edwards MR, Parker R, et al., 2017, Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways, PLoS Pathogens, Vol: 13, ISSN: 1553-7366
Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1β and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myleoid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb.
Khara JS, Obuobi S, Wang Y, et al., 2017, Disruption of drug-resistant biofilms using de novo designed short α-helicalantimicrobial peptides with idealized facial amphiphilicity, Acta Biomaterialia, Vol: 57, Pages: 103-114, ISSN: 1878-7568
The escalating threat of antimicrobial resistance has increased pressure to develop novel therapeutic strategies to tackle drug-resistant infections. Antimicrobial peptides have emerged as a promising class of therapeutics for various systemic and topical clinical applications. In this study, the de novo design of α-helical peptides with idealized facial amphiphilicities, based on an understanding of the pertinent features of protein secondary structures, is presented. Synthetic amphiphiles composed of the backbone sequence (X1Y1Y2X2)n, where X1 and X2 are hydrophobic residues (Leu or Ile or Trp), Y1 and Y2 are cationic residues (Lys), and n is the number repeat units (2 or 2.5 or 3), demonstrated potent broad-spectrum antimicrobial activities against clinical isolates of drug-susceptible and multi-drug resistant bacteria. Live-cell imaging revealed that the most selective peptide, (LKKL)3, promoted rapid permeabilization of bacterial membranes. Importantly, (LKKL)3 not only suppressed biofilm growth, but effectively disrupted mature biofilms after only 2 h of treatment. The peptides (LKKL)3 and (WKKW)3 suppressed the production of LPS-induced pro-inflammatory mediators to levels of unstimulated controls at low micromolar concentrations. Thus, the rational design strategies proposed herein can be implemented to develop potent, selective and multifunctional α-helical peptides to eradicate drug-resistant biofilm-associated infections.
Khara JS, Priestman M, Uhia I, et al., 2016, Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability, Journal of Antimicrobial Chemotherapy, Vol: 71, Pages: 2181-2191, ISSN: 1460-2091
Objectives The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities.Methods Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy.Results Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all D-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified L-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm.Conclusions Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.
von Both U, Levin M, Kaforou M, et al., 2015, Understanding immune protection against tuberculosis using RNA expression profiling., Vaccine, Vol: 33, Pages: 5289-5293, ISSN: 1873-2518
A major limitation in the development and testing of new tuberculosis (TB) vaccines is the current inadequate understanding of the nature of the immune response required for protection against either infection with Mycobacterium tuberculosis (MTB) or progression to disease. Genome wide RNA expression analysis has provided a new tool with which to study the inflammatory and immunological response to mycobacteria. To explore how currently available transcriptomic data might be used to understand the basis of protective immunity to MTB, we analysed and reviewed published RNA expression studies to (1) identify a “susceptible” immune response in patients with acquired defects in the interferon gamma pathway; (2) identify the “failing” transcriptomic response in patients with TB as compared with latent TB infection (LTBI); and (3) identify elements of the “protective” response in healthy latently infected and healthy uninfected individuals.Abbreviations TB, tuberculosis; MTB, Mycobacterium tuberculosis; IFN-γ, interferon-gamma; PBMC, peripheral blood mononuclear cells; MSMD, Mendelian susceptibility to mycobacterial disease; BCG, bacille Calmette–Guerin; LTBI, latent tuberculosis infectionKeywords Transcriptomics; RNA expression profiling; Tuberculosis; Vaccines; Interferon-γ; Type I interferon
Schiebler M, Brown K, Hegyi K, et al., 2014, Functional drug screening reveals anticonvulsants as enhancers of mTOR‐independent autophagic killing of Mycobacterium tuberculosis through inositol depletion, EMBO Molecular Medicine, Vol: 7, Pages: 127-139, ISSN: 1757-4676
Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell‐based screening of FDA‐approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug‐resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR‐independent pathway controlled by cellular depletion of myo‐inositol. While strain‐specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug‐resistant mycobacterial infection.
Khara JS, Wang Y, Ke X-Y, et al., 2014, Anti-mycobacterial activities of synthetic cationic alpha-helical peptides and their synergism with rifampicin, BIOMATERIALS, Vol: 35, Pages: 2032-2038, ISSN: 0142-9612
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Renna M, Schaffner C, Brown K, et al., 2011, Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection, Journal of Clinical Investigation, Vol: 121, Pages: 3554-3563, ISSN: 1558-8238
Azithromycin is a potent macrolide antibiotic with poorly understood antiinflammatory properties. Long-term use of azithromycin in patients with chronic inflammatory lung diseases, such as cystic fibrosis (CF), results in improved outcomes. Paradoxically, a recent study reported that azithromycin use in patients with CF is associated with increased infection with nontuberculous mycobacteria (NTM). Here, we confirm that long-term azithromycin use by adults with CF is associated with the development of infection with NTM, particularly the multi-drug-resistant species Mycobacterium abscessus, and identify an underlying mechanism. We found that in primary human macrophages, concentrations of azithromycin achieved during therapeutic dosing blocked autophagosome clearance by preventing lysosomal acidification, thereby impairing autophagic and phagosomal degradation. As a consequence, azithromycin treatment inhibited intracellular killing of mycobacteria within macrophages and resulted in chronic infection with NTM in mice. Our findings emphasize the essential role for autophagy in the host response to infection with NTM, reveal why chronic use of azithromycin may predispose to mycobacterial disease, and highlight the dangers of inadvertent pharmacological blockade of autophagy in patients at risk of infection with drug-resistant pathogens.
Newton SM, Martineau AR, Kampmann B, 2011, A functional whole blood assay to measure immune responses to mycobacteria, using reporter-gene tagged BCG or Mycobacterium tuberculosis, J.Vis. Exp., Vol: 55
Martineau AR, Leandro ACCS, Anderson ST, et al., 2010, Association between Gc genotype and susceptibility to TB is dependent on vitamin D status, EUROPEAN RESPIRATORY JOURNAL, Vol: 35, Pages: 1106-1112, ISSN: 0903-1936
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Wilkinson KA, Newton SM, Stewart GR, et al., 2009, Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis, BMC MICROBIOLOGY, Vol: 9, ISSN: 1471-2180
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Coussens A, Timms PM, Boucher BJ, et al., 2008, 1alpha,25-dihydroxyvitamin D inhibits matrix metalloproteinases induced by Mycobacterium tuberculosis infection, Immunology, Vol: Epub ahead of print
Newton SM, Brent AJ, Anderson S, et al., 2008, Paediatric tuberculosis, LANCET INFECTIOUS DISEASES, Vol: 8, Pages: 498-510, ISSN: 1473-3099
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Newton SM, Mackie SL, Martineau AR, et al., 2008, Reduction of chemokine secretion in response to mycobacteria in infliximab-treated patients, CLINICAL AND VACCINE IMMUNOLOGY, Vol: 15, Pages: 506-512, ISSN: 1556-6811
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Martineau AR, Wilkinson KA, Newton SM, et al., 2007, IFN-gamma- and TNF-independent vitamin D-inducible human suppression of mycobacteria: the role of cathelicidin LL-37 (vol 178, pg 7190, 2007), JOURNAL OF IMMUNOLOGY, Vol: 179, Pages: 8569-8570, ISSN: 0022-1767
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Martineau AR, Wilkinson KA, Newton SM, et al., 2007, IFN-γ- and TNF-independent vitamin D-inducible human suppression of mycobacteria: The role of cathelicidin LL-37 (Journal of Immunology (2007) 178 (7190-7198)), Journal of Immunology, Vol: 179, Pages: 8569-8570, ISSN: 0022-1767
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Martineau AR, Wilkinson RJ, Wilkinson KA, et al., 2007, A single dose of vitamin D enhances immunity to mycobacteria, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 176, Pages: 208-213, ISSN: 1073-449X
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Martineau AR, Newton SM, Wilkinson KA, et al., 2007, Neutrophil-mediated innate immune resistance to mycobacteria, JOURNAL OF CLINICAL INVESTIGATION, Vol: 117, Pages: 1988-1994, ISSN: 0021-9738
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Martineau AR, Wilkinson KA, Newton SM, et al., 2007, IFN-γ- and TNF-independent vitamin D-inducible human suppression of mycobacteria: The role of cathelicidin LL-37, Journal of Immunology, Vol: 178, Pages: 7190-7198, ISSN: 0022-1767
Vitamin D deficiency is associated with susceptibility to tuberculosis, and its biologically active metabolite, 1α,25 dihydroxyvitamin D3 (1α,25(OH)2D3), has pleiotropic immune effects. The mechanisms by which 1α,25(OH)2D3 protects against tuberculosis are incompletely understood. 1α,25(OH)2D 3 reduced the growth of mycobacteria in infected human PBMC cultures in a dose-dependent fashion. Coculture with agonists or antagonists of the membrane or nuclear vitamin D receptors indicated that these effects were primarily mediated by the nuclear vitamin D receptors. 1α,25(OH) 2D3 reduced transcription and secretion of protective IFN-γ, IL-12p40, and TNF in infected PBMC and macrophages, indicating that 1α,25(OH)2D3 does not mediate protection via these cytokines. Although NOS2A was up-regulated by 1α,25(OH)2D 3, inhibition of NO formation marginally affected the suppressive effect of 1α,25(OH)2D3 on bacillus Calmette Guérin in infected cells. By contrast, 1α,25(OH)2D 3 strongly up-regulated the cathelicidin hCAP-18 gene, and some hCAP-18 polypeptide colocalized with CD14 in 1α,25(OH)2D 3 stimulated PBMC, although no detectable LL-37 peptide was found in supernatants from similar 1α,25(OH)2D3-stimulated PBMC cultures. A total of 200 μg/ml of the active peptide LL-37, in turn, reduced the growth of Mycobacterium tuberculosis in culture by 75.7%. These findings suggest that vitamin D contributes to protection against TB by "nonclassical" mechanisms that include the induction of antimicrobial peptides. Copyright © 2007 by The American Association of Immunologists, Inc.
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