Characterizing Mycobacterium abscessus complex (MABSC) biofilms under host-relevant conditions is essential to the design of informed therapeutic strategies targeted to this persistent, drug-tolerant, population of extracellular bacilli. Using synthetic cystic fibrosis medium (SCFM) which we previously reported to closely mimic the conditions encountered by MABSC in actual cystic fibrosis (CF) sputum and a new model of biofilm formation, we show that MABSC biofilms formed under these conditions are substantially different from previously reported biofilms grown in standard laboratory media in terms of their composition, gene expression profile and stress response. Extracellular DNA (eDNA), mannose-and glucose-containing glycans and phospholipids, rather than proteins and mycolic acids, were revealed as key extracellular matrix (ECM) constituents holding clusters of bacilli together. None of the environmental cues previously reported to impact biofilm development had any significant effect on SCFM-grown biofilms, most likely reflecting the fact that SCFM is a nutrient-rich environment in which MABSC finds a variety of ways of coping with stresses. Finally, molecular determinants were identified that may represent attractive new targets for the development of adjunct therapeutics targeting MABSC biofilms in persons with CF.

Mice infected for 60 days with Mycobacterium tuberculosis were treated with aerosolized XCL1-targeting small interfering RNA (siRNA) to induce local and transient suppression of XCL1/lymphotactin (an important chemokine in tuberculoid granuloma formation). The local pulmonary siRNA therapy resulted in a 50% decrease in the total amount of xcl1 gene transcripts at 3 days, and 40 to 50% protein suppression 3 and 5 days after treatment. Reduced XCL1 expression in the lungs was associated with decreased numbers of T lymphocytes, reduction in the IFN-g response, disorganized granulomatous lesions, and higher fibrosis when compared with control mice treated with either PBS or nontargeting siRNA. This indicates that a transient but strong modulation of the production of XCL1 in the lungs has a significant effect on the influx of IFN-g-secreting T cells, as well as local pathology, but without significantly altering containment of the infection.Keywords: tuberculosis; small interfering RNA; lymphotactin; XCL1; aerosol deliveryUp to now, in vivo studies addressing the role of specific components of the immune response during chronic infection with Mycobacterium tuberculosis were limited to the use of genetargeted knockout mice or systemic antibody and drug delivery. Although these methods provided important information in regards to tuberculosis infection, they do not allow for targeted examination of the lung-unique environment. Furthermore, gene knockout mice have the deficiency from the onset, thus not allowing more temporal manipulation of the immune response. For that reason, here, we tried an innovative approach in which newly developed immunotherapeutic molecules were applied directly to the lungs. Specifically, we transiently changed the lung immune environment by delivery of small interfering RNA (siRNA) transcripts.siRNA is a technology being used to evaluate the function of a variety of genes by transient silencing of mRNA expression. This new technology has been used as a therapeutic procedure to treat a variety of genetic, viral, and cancer-related diseases (1). It has demonstrated therapeutic benefits after both local and systemic administration into subcutaneous tissue, muscle, eye, and the central nervous system (2, 3). The major challenge of using siRNA as an immunotherapy is to deliver it into tissues and then into the cytoplasm of cells. Exceptions to this are the mucosal tissues and the lung. In these tissues, it has been demonstrated that siRNA uptake is extremely efficient and occurs even in the absence of transfection reagents (4-7). We took advantage of this, and developed a procedure to transiently block expression of proteins by using a noninvasive procedure for intrapulmonary delivery of aerosolized siRNA. Using this approach we studied changes in the immunopathology in mice chronically infected with M. tuberculosis.Tuberculosis is a global problem caused by infection with the M. tuberculosis bacilli. At the present time, one third of the world population has been exposed to this bacillus, and ...

In this study we demonstrate that it is possible to shift the immune system during a chronic infection with Mycobacterium tuberculosis. TGFβ and IL10 cytokines inhibit the Th1 response during chronic pulmonary infection with M. tuberculosis. We show that intrapulmonary delivery of siRNA targeting TGFβ1 is able to reduce the pulmonary bacillary load in mice chronically infected with M. tuberculosis: an effect that appears to be partly dependent on IL10 expression. To demonstrate this, we induced gene silencing of tgfβ1 in the lungs of wild type and IL10 knockout mice using a non-invasive aerosolized intrapulmonary delivery of siRNA targeting TGFβ1. Five days after the last treatment with siRNA, the levels of tgfb1 transcripts and TGFβ1 protein were reduced when compared with control groups treated with RNase-free water or non-targeting siRNA. Mice treated with siRNA also had increased expression of the antimicrobial mediators (NO and iNOS) which effectively reduced the bacterial load by 0.17 and 0.47 log(10) in C57BL/6 and IL-10 KO mice respectively when compared with their respective control mice. More importantly, the bacterial load in siRNA treated IL-10 KO mice four weeks after the last treatment remained 0.32 log(10) lower than in control mice.

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A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis , is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS . OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.

Background Mycobacterium abscessus causes chronic pulmonary infections. Owing to its resistance to most classes of antibiotics, treatment is complex and cure rates are only 45%. Tigecycline is active against M. abscessus, but severe toxicity and the need for IV administration limit its use. Objectives To assess the potential of inhaled tigecycline as a treatment for M. abscessus pulmonary disease, by measuring its efficacy in a mouse model of chronic M. abscessus pulmonary disease, establishing the intracellular activity of tigecycline against M. abscessus in human macrophages and measuring the activity of tigecycline in the sputum of cystic fibrosis patients. Methods We infected GM-CSF knockout mice with M. abscessus by intrapulmonary aerosol. Infected mice were treated with tigecycline in 0.25, 1.25 and 2.5 mg doses, by inhalation, or untreated, for 28 days. Tigecycline was added to human peripheral blood-derived macrophages infected with M. abscessus to assess its intracellular activity. We performed a time–kill kinetics experiment of tigecycline against M. abscessus with and without sputum of cystic fibrosis patients. Results Inhaled tigecycline proved highly effective against M. abscessus in GM-CSF knockout mice. The effect was dose dependent. Tigecycline showed potent activity against M. abscessus in macrophages and retained most of its activity in the presence of sputum of cystic fibrosis patients. Conclusions Inhaled tigecycline may represent a viable treatment option for M. abscessus pulmonary disease, where treatment outcomes are currently very poor. A stable and safe formulation is required to proceed to further pharmacodynamic studies and ultimately clinical trials.

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SummaryInhaled therapies in the form of drugs or vaccines for tuberculosis treatment were reported about a decade ago. Experts around the world met to discuss the scientific progress in inhaled therapies at the international symposium "Optimization of inhaled Tuberculosis therapies and implications for host-pathogen interactions" held in New Delhi, India on November 3-5, 2009. The meeting was organized by the Central Drug Research Institute (CDRI) Lucknow, India. The lung is the main route for infection with Mycobacterium tuberculosis bacilli and the primary site of reactivation of latent disease. The only available vaccine BCG is relatively ineffective at preventing tuberculosis disease and current therapy requires prolonged treatment with drugs which results in low patient compliance. Consequently, there is a need to design new vaccines and therapies for this disease. Recently there has been increased interest in the development of inhaled formulations to deliver anti-mycobacterial drugs and vaccines directly to the lung and many of these therapies are designed to target lung macrophages and dendritic cells. However, the development of effective inhaled therapies requires an understanding of the unique function and immunosuppressive environment of the lung which is driven, in part, by alveolar macrophages and dendritic cells. In this review, we will discuss the role of alveolar macrophages and dendritic cells in the host immune response to M. tuberculosis infection and the ways in which inhaled therapies might enhance the anti-microbial response of phagocytes and boost pulmonary immunity.Keywords: Tuberculosis; Macrophage; Dendritic cell; Inhaled therapy stimulate innate bactericidal responses and/or antigen presenting functions more efficiently and will ameliorate drug toxicity due to reduction in dose/duration of treatment.12 However, the development of successful inhaled therapies in general will depend on the confounding characteristics of the lungs, in addition to the multiple variables added by alterations in its structure and damage inflicted by the inflammatory process, and needs to take into account factors such as aeration of the lungs, deposition and lung function, formation of biofilms and resistance in the face of treatment. [12] and [13]