Deletion of cftr Leads to an Excessive Neutrophilic Response and Defective Tissue Repair in a Zebrafish Model of Sterile Inflammation.

Inflammation-related progressive lung destruction is the leading causes of premature death in cystic fibrosis (CF), a genetic disorder caused by a defective cystic fibrosis transmembrane conductance regulator (CFTR). However, therapeutic targeting of inflammation has been hampered by a lack of understanding of the links between a dysfunctional CFTR and the deleterious innate immune response in CF. Herein, we used a CFTR-depleted zebrafish larva, as an innovative in vivo vertebrate model, to understand how CFTR dysfunction leads to abnormal inflammatory status in CF. We show that impaired CFTR-mediated inflammation correlates with an exuberant neutrophilic response after injury: CF zebrafish exhibit enhanced and sustained accumulation of neutrophils at wounds. Excessive epithelial oxidative responses drive enhanced neutrophil recruitment towards wounds. Persistence of neutrophils at inflamed sites is associated with impaired reverse migration of neutrophils and reduction in neutrophil apoptosis. As a consequence, the increased number of neutrophils at wound sites causes tissue damage and abnormal tissue repair. Importantly, the molecule Tanshinone IIA successfully accelerates inflammation resolution and improves tissue repair in CF animal. Our findings bring important new understanding of the mechanisms underlying the inflammatory pathology in CF, which could be addressed therapeutically to prevent inflammatory lung damage in CF patients with potential improvements in disease outcomes

The Diverse Cellular and Animal Models to Decipher the Physiopathological Traits of Mycobacterium abscessus Infection

Mycobacterium abscessus represents an important respiratory pathogen among the rapidly-growing non-tuberculous mycobacteria. Infections caused by M. abscessus are increasingly found in cystic fibrosis (CF) patients and are often refractory to antibiotic therapy. The underlying immunopathological mechanisms of pathogenesis remain largely unknown. A major reason for the poor advances in M. abscessus research has been a lack of adequate models to study the acute and chronic stages of the disease leading to delayed progress of evaluation of therapeutic efficacy of potentially active antibiotics. However, the recent development of cellular models led to new insights in the interplay between M. abscessus with host macrophages as well as with amoebae, proposed to represent the environmental host and reservoir for non-tuberculous mycobacteria. The zebrafish embryo has also appeared as a useful alternative to more traditional models as it recapitulates the vertebrate immune system and, due to its optical transparency, allows a spatio-temporal visualization of the infection process in a living animal. More sophisticated immunocompromised mice have also been exploited recently to dissect the immune and inflammatory responses to M. abscessus. Herein, we will discuss the limitations, advantages and potential offered by these various models to study the pathophysiology of M. abscessus infection and to assess the preclinical efficacy of compounds active against this emerging human pathogen

Inhibition of the β-lactamase BlaMab by avibactam improves the in vitro and in vivo efficacy of imipenem against mycobacterium abscessus

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M. abscessus produces the broad-spectrum β-lactamase BlaMab. We determine whether inhibition of BlaMab by avibactam improves the activity of imipenem against M. abscessus. The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of BlaMab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (<0.1% survival), with 3.2- and 4.3-log10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of BlaMab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding BlaMab was induced (20-fold) in the infected macrophages. Inhibition of BlaMab by avibactam improved the efficacy of imipenem against M. abscessus in vitro, in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of BlaMab, since the production of the β-lactamase is inducible in macrophages

Polymeric nanobiotics as a novel treatment for mycobacterial infections.

Mycobacterium tuberculosis (Mtb) remains a major challenge to global health, made worse by the spread of multi-drug resistance. Currently, the efficacy and safety of treatment is limited by difficulties in achieving and sustaining adequate tissue antibiotic concentrations while limiting systemic drug exposure to tolerable levels. Here we show that nanoparticles generated from a polymer-antibiotic conjugate ('nanobiotics') deliver sustained release of active drug upon hydrolysis in acidic environments, found within Mtb-infected macrophages and granulomas, and can, by encapsulation of a second antibiotic, provide a mechanism of synchronous drug delivery. Nanobiotics are avidly taken up by infected macrophages, enhance killing of intracellular Mtb, and are efficiently delivered to granulomas and extracellular mycobacterial cords in vivo in an infected zebrafish model. We demonstrate that isoniazid (INH)-derived nanobiotics, alone or with additional encapsulation of clofazimine (CFZ), enhance killing of mycobacteria in vitro and in infected zebrafish, supporting the use of nanobiotics for Mtb therapy and indicating that nanoparticles generated from polymer-small molecule conjugates might provide a more general solution to delivering co-ordinated combination chemotherapy.Rosetrees Trust Interdisciplinary Prize 2015 Wellcome Trust awards 107032/Z/15/Z and 10/H0305/55 NIHR Cambridge Biomedical Research Centre Award MRC AMR Theme award MR/N02995X/1 Marie-Curie IF CFZEBRA 75197

CFTR protects against Mycobacterium abscessus infection by fine-tuning host oxidative defenses

Infection by rapidly growing Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF), a genetic disease caused by a defective CF transmembrane conductance regulator (CFTR). However, the potential link between a dysfunctional CFTR and vulnerability to M. abscessus infection remains unknown. Herein, we exploit a CFTR-depleted zebrafish model, recapitulating CF immuno-pathogenesis, to study the contribution of CFTR in innate immunity against M. abscessus infection. Loss of CFTR increases susceptibility to infection through impaired NADPH oxidase-dependent restriction of intracellular growth and reduced neutrophil chemotaxis, which together compromise granuloma formation and integrity. As a consequence, extracellular multiplication of M. abscessus expands rapidly, inducing abscess formation and causing lethal infections. Because these phenotypes are not observed with other mycobacteria, our findings highlight the crucial and specific role of CFTR in the immune control of M. abscessus by mounting effective oxidative responses

Neutrophil killing of Mycobacterium abscessus by intra- and extracellular mechanisms

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, are increasingly present in soft tissue infections and chronic lung diseases, including cystic fibrosis, and infections are characterized by growth in neutrophil-rich environments. M. abscessus is observed as two distinct smooth and rough morphotypes. The environmental smooth morphotype initiates infection and has a relatively limited ability to activate neutrophils. The rough morphotype has increased virulence and immunogenicity. However, the neutrophil response to the rough morphotype has not been explored. Killing of the smooth and rough strains, including cystic fibrosis clinical isolates, was equivalent. Neutrophil uptake of M. abscessus was similar between morphotypes. Mechanistically, both rough and smooth morphotypes enhanced neutrophil reactive oxygen species generation but inhibition of NADPH oxidase activity did not affect M. abscessus viability. However, inhibition of phagocytosis and extracellular traps reduced killing of the smooth morphotype with lesser effects against the rough morphotype. Neutrophils treated with M. abscessus released a heat-labile mycobactericidal activity against the rough morphotype, but the activity was heat-tolerant against the smooth morphotype. Overall, M. abscessus stimulates ineffective neutrophil reactive oxygen species generation, and key mechanisms differ in killing of the smooth (phagocytosis-dependent, extracellular traps, and heat-tolerant secreted factor) and rough (extracellular traps and a heat-labile secreted factor) morphotypes. These studies represent an essential advancement in understanding the host response to M. abscessus, and help explain the recalcitrance of infection

Zebrafish Infection: From Pathogenesis to Cell Biology

The study of host–pathogen interactions has illuminated fundamental research avenues in both infection and cell biology. Zebrafish (Danio rerio) larvae are genetically tractable, optically accessible, and present a fully functional innate immune system with macrophages and neutrophils that mimic their mammalian counterparts. A wide variety of pathogenic bacteria have been investigated using zebrafish models, providing unprecedented resolution of the cellular response to infection in vivo. In this review, we illustrate how zebrafish models have contributed to our understanding of cellular microbiology by providing an in vivo platform to study host–pathogen interactions from the single cell to whole animal level. We also highlight discoveries made from zebrafish infection that hold great promise for translation into novel therapies for humans

The inflammatory chemokine Cxcl18b exerts neutrophil-specific chemotaxis via the promiscuous chemokine receptor Cxcr2 in zebrafish

Cxcl18b is a chemokine found in zebrafish and in other piscine and amphibian species. Cxcl18b is a reliable inflammatory marker; however, its function is yet to be elucidated. Here, we found that Cxcl18b is chemotactic towards neutrophils, similarly to Cxcl8a/Interleukin-8, the best characterised neutrophil chemoattractant in humans and teleosts. Like Cxcl8a, Cxcl18b-dependent recruitment required the chemokine receptor Cxcr2, while it was unaffected by depletion of the other two neutrophil receptors cxcr1 and cxcr4b. To visualise cxcl18b induction, we generated a Tg(cxcl18b:eGFP) reporter line. The transgene is induced locally upon bacterial infection with the fish pathogen Mycobacterium marinum, but strikingly is not directly expressed by infected cells. Instead, cxcl18b is induced by non-phagocytic uninfected cells that compose the stroma of the granulomas, typical inflammatory lesions formed upon mycobacterial infections. Together, these results suggest that Cxcl18b might be an important contributor to neutrophil chemotaxis in the inflammatory microenvironment and indicate that the zebrafish model could be explored to further investigate in vivo the biological relevance of different Cxcl8-like chemokine lineages

Polymeric nanobiotics as a novel treatment for mycobacterial infections

Mycobacterium tuberculosis (Mtb) remains a major challenge to global health, made worse by the spread of multi-drug resistance. Currently, the efficacy and safety of treatment is limited by difficulties in achieving and sustaining adequate tissue antibiotic concentrations while limiting systemic drug exposure to tolerable levels. Here we show that nanoparticles generated from a polymer-antibiotic conjugate (‘nanobiotics’) deliver sustained release of active drug upon hydrolysis in acidic environments, found within Mtb-infected macrophages and granulomas, and can, by encapsulation of a second antibiotic, provide a mechanism of synchronous drug delivery. Nanobiotics are avidly taken up by infected macrophages, enhance killing of intracellular Mtb, and are efficiently delivered to granulomas and extracellular mycobacterial cords in vivo in an infected zebrafish model. We demonstrate that isoniazid (INH)-derived nanobiotics, alone or with additional encapsulation of clofazimine (CFZ), enhance killing of mycobacteria in vitro and in infected zebrafish, supporting the use of nanobiotics for Mtb therapy and indicating that nanoparticles generated from polymer-small molecule conjugates might provide a more general solution to delivering co-ordinated combination chemotherapy

Aggregation of nontuberculous mycobacteria is regulated by carbon-nitrogen balance

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that colonize household water systems and cause chronic lung infections in susceptible patients. The ability of NTM to form surface-attached biofilms in the nonhost environment and corded aggregates in vivo is important to their ability to persist in both contexts. Underlying the development of these multicellular structures is the capacity of mycobacterial cells to adhere to one another. Unlike most other bacteria, NTM spontaneously and constitutively aggregate in vitro, hindering our ability to understand the transition between planktonic and aggregated cells. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulate after ∼3 days of growth. By providing selective pressure for bacteria that disperse earlier, we isolated a strain with two mutations in the oligopeptide permease operon (opp). A mutant lacking the opp operon (Δopp) disperses earlier than wild type (WT) due to a defect in nutrient uptake. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, but under conditions of low available carbon relative to available nitrogen, M. smegmatis grows as planktonic cells. By adjusting carbon and nitrogen sources in defined medium, we tuned the cellular C/N ratio such that M. smegmatis grows either as aggregates or as planktonic cells. C/N-mediated aggregation regulation is widespread among NTM with the possible exception of rough-colony Mycobacterium abscessus isolates. Altogether, we show that NTM aggregation is a controlled process that is governed by the relative availability of carbon and nitrogen for metabolism