Tribbles1 is host protective during in vivo mycobacterial infection.

Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production, and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown/knockout exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of proinflammatory factors il-1ß and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.

Drug repositioning identifies histone deacetylase inhibitors that promote innate immunity in non-tuberculous mycobacterial infection.

Non-tuberculosis infections in immunocompromised patients represent a cause for concern, given the increased risks of infection, and limited treatments available. Herein, we report that molecules for binding to the catalytic site of histone deacetylase (HDAC) inhibit its activity, thus increasing the innate immune response against environmental mycobacteria. The action of HDAC inhibitors (iHDACs) was explored in a model of type II pneumocytes and macrophages infection by Mycobacterium aurum. The results show that the use of 1,3-diphenylurea increases the expression of the TLR-4 in M. aurum infected MDMs, as well as the production of defb4, IL-1ß, IL-12, and IL-6. Moreover, we observed that aminoacetanilide upregulates the expression of TLR-4 together with TLR-9, defb4, CAMP, RNase 6, RNase 7, IL-1ß, IL-12, and IL-6 in T2P. Results conclude that the tested iHDACs selectively modulate the expression of cytokines and antimicrobial peptides that are associated with reduction of non-tuberculous mycobacteria infection.

Attenuated tuberculin skin test responses associated with Mycobacterium intracellulare sputum colonization in an adolescent TB prevalence survey in Western Kenya.

INTRODUCTION: Exposure to Non-tuberculous Mycobacteria (NTM) varies regionally and may partly explain the disparate outcomes of BCG vaccination and tuberculosis (TB) susceptibility. METHODS: We examined NTM sputum colonization, associations with clinical characteristics, and tuberculin skin test (TST) responses in an adolescent TB prevalence survey. RESULTS: Among 5004 adolescents screened, 2281 (45.5 %) were evaluated further. TB and NTM prevalence rates were 0.3 % and 8.0 %, respectively. Among 418 NTM isolates, 103 were unidentifiable, and 315 (75 %) comprised 15 species, the most frequent being M. intracellulare (MAC) (108, 26 %), M. scrofulaceum (96, 23 %) and M. fortuitum (51, 12 %). "NTM colonized" adolescents had less frequent chronic cough and night sweats (adjusted odds ratio [aOR] 0.62, 95 % confidence interval [CI] 0.44-0.87and aOR 0.61, CI 0.42-0.89 respectively), and lower TST induration (median 11 mm (interquartile range [IQR] 0-16) vs 13 mm (IQR 6-17; p = 0.006)) when compared to "NTM not colonized" participants. MAC, but not M. scrofulaceum or M. fortuitum, was associated with decreased TST induration (median 7.5 mm (IQR 0-15) vs 13 mm (IQR 6-17) among "MAC colonized" vs "not colonized", p = 0.001). CONCLUSION: We observed high NTM prevalence rates with species-specific associations with TST induration, consistent with a model of species-dependent heterologous immunity among mycobacteria.

Transcriptome profiles of macrophages upon infection by morphotypic smooth and rough variants of Mycobacterium abscessus.

Mycobacterium abscessus (Mab) infection can be deadly in patients with chronic lung diseases like cystic fibrosis (CF). In vitro and in vivo, Mab may adopt a smooth (S) or rough (R) morphotype, the latter linked to more severe disease conditions. In vitro studies revealed differences in pathogenicity and immune response to S and R morphotypes. We propose that in vivo both morphotypes exist and may transiently switch depending on the environment, having important pathogenic and immunologic consequences. This can be modeled by morphotypic S and R variants of Mab selected based on in vitro growth conditions. Here, we report the first analysis of early transcriptional events in mouse bone marrow derived macrophages (BMDMs) upon infection with media-selected interchangeable Mab-S and Mab-R morphotypes. The early transcriptional events after infection with both morphotypes showed considerable overlap of the pro-inflammatory genes that were differentially regulated compared to the uninfected macrophages. We also observed signature genes significantly differentially regulated in macrophages during infection of media-selected morphotypic Mab-S and Mab-R variants. In conclusion, media-selected Mab-S and Mab-R behave in a similar fashion to stable S and R types with respect to pathogenesis and immune response, serving as a useful model for environmentally influenced morphotype selection.

Immunological and microbial shifts in the aging rhesus macaque lung during nontuberculous mycobacterial infection.

Nontuberculous mycobacteria (NTM) are environmentally ubiquitous organisms that predominately cause NTM pulmonary disease (NTMPD) in individuals over the age of 65. The incidence of NTMPD has increased in the U.S., exceeding that of Mycobacterium tuberculosis. However, the mechanisms leading to higher susceptibility and severity of NTMPD with aging are poorly defined in part due to the lack of animal models that accurately recapitulate human disease. Here, we compared bacterial load, microbial communities, and host responses longitudinally between three young (two female and one male) and two aged (two female) rhesus macaques inoculated with Mycobacterium avium subsp. hominissuis (MAH) in the right caudal lobe. Unilateral infection resulted in a low bacterial load in both young and aged animals confined to the infected side. Although a robust inflammatory response was only observed in the inoculated lung, immune cell infiltration and antigen-specific T cells were detected in both lungs. Computed tomography, gross pathology, and histopathology revealed increased disease severity and persistence of bacterial DNA in aged animals. Additional analyses showed the translocation of gut and oral-pharyngeal bacterial DNA into the lower respiratory microbiome. Finally, single-cell RNA sequencing revealed a heightened inflammatory response to MAH infection by alveolar macrophages in aged animals. These data are consistent with the model that increased disease severity in the aged is mediated by a dysregulated macrophage response that may be sustained through persistent antigen presence. IMPORTANCE: Nontuberculous mycobacteria (NTM) are emerging as pathogens of high consequence, as cases of NTM pulmonary disease (NTMPD) have exceeded those of Mycobacterium tuberculosis. NTMPD can be debilitating, particularly in patients over 65 years of age, as it causes chronic cough and fatigue requiring prolonged treatments with antibiotics. The underlying mechanisms of this increased disease severity with age are poorly understood, hampering the development of therapeutics and vaccines. Here, we use a rhesus macaque model to investigate the impact of age on host-NTM interactions. This work shows that aging is associated with increased disease severity and bacterial persistence in aged rhesus macaques, thus providing a preclinical model to develop and test novel therapeutics and interventions.

Impact of IL-1ß on lung pathology caused by Mycobacterium abscessus infection and its association with IL-17 production.

Mycobacterium abscessus (MAB), a non-tuberculous mycobacterium (NTM), causes chronic pulmonary inflammation in humans. The NLRP3 inflammasome is a multi-protein complex that triggers IL-1ß maturation and pyroptosis through the cleavage of caspase-1. In this study, we investigated the roles of NLRP3 and IL-1ß in the host's defense against MAB. The IL-1ß production by MAB was completely abolished in NLRP3, but not NLRC4, deficient macrophages. The NLRP3 inflammasome components, which are ASC and caspase-1 were also found to be essential for IL-1ß production in response to MAB. NLRP3 and IL-1ß deficiency did not affect the intracellular growth of MAB in macrophages, and the bacterial burden in lungs of NLRP3- and IL-1ß-deficient mice was also comparable to the burden observed in WT mice. In contrast, IL-1ß deficiency ameliorated lung pathology in MAB-infected mice. Notably, the lung homogenates of IL-1ß-deficient mice had reduced levels of IL-17, but not IFN-γ and IL-4 when compared with WT counterparts. Furthermore, in vitro co-culture analysis showed that IL-1ß signaling was essential for IL-17 production in response to MAB. Finally, we observed that the anti-IL-17 antibody administration moderately mitigated MAB-induced lung pathology. These findings indicated that IL-1ß production contribute to MAB-induced lung pathology via the elevation of IL-17 production.

Colony morphotype governs innate and adaptive pulmonary immune responses to Mycobacterium abscessus infection in C3HeB/FeJ mice.

Mycobacterium abscessus is an emerging pathogen that causes chronic pulmonary infection. Treatment is challenging owing in part to our incomplete understanding of M. abscessus virulence mechanisms that enable pathogen persistence, such as the differing pathogenicity of M. abscessus smooth (S) and rough (R) colony morphotype. While R M. abscessus is associated with chronic infection and worse patient outcomes, it is unknown how immune responses to S and R M. abscessus differ in an acute pulmonary infection setting. In this study, immunological outcomes of M. abscessus infection with S and R morphotypes were examined in an immune-competent C3HeB/FeJ murine model. R M. abscessus infection was associated with the rapid production of inflammatory chemokines and recruitment of activated, MHC-II+ Ly6C+ macrophages to lungs and mediastinal LN (mLN). While both S and R M. abscessus increased T helper 1 (Th1) phenotype T cells in the lung, this was markedly delayed in mice infected with S M. abscessus. However, histopathological involvement and bacterial clearance were similar regardless of colony morphotype. These results demonstrate the importance of M. abscessus colony morphotype in shaping the development of pulmonary immune responses to M. abscessus, which further informs our understanding of M. abscessus host-pathogen interactions.

In vitro stimulation with nontuberculous mycobacteria induced a stronger cytokine response in leukocytes isolated from individuals with latent tuberculosis compared to those isolated from active tuberculosis or cystic fibrosis patients.

Mycobacterium tuberculosis and opportunistic environmental non-tuberculous mycobacteria (NTM) can cause severe infection. Why latent tuberculosis infection advances to active disease, and why some individuals with cystic fibrosis (CF) develop pulmonary infections with NTM is still poorly understood. The aim of this study was to investigate the effector function of peripheral blood mononuclear cells (PBMC) from individuals with active or latent tuberculosis, individuals with CF with or without pulmonary NTM-infection and healthy controls, by measuring cytokine response to in vitro stimulation with different species of NTMs. The cytokine concentrations of IL-17A, IL-22, IL-23, IL-10, IL12p70 and IFN-γ were measured in PBMC-culture supernatants after stimulation with NTMs. PBMCs from individuals with latent tuberculosis infection showed strong IL-17A, IL-22, and IFN-γ responses compared to individuals with active tuberculosis or CF. IL-10 production was low in both tuberculosis groups compared to the CF groups and controls. This study suggests that IL-17A and IL-22 might be important to keep tuberculosis in a latent phase and that individuals with CF with an ongoing NTM infection seem to have a low cytokine response.

E3 ubiquitin ligase CBLB regulates innate immune responses and bacterial dissemination during nontuberculous mycobacteria infection.

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens causing pulmonary infection to fatal disseminated disease. NTM infections are steadily increasing in children and adults, and immune-compromised individuals are at a greater risk of fatal infections. The NTM disease's adverse pathology and resistance to antibiotics have further worsened the therapeutic measures. Innate immune regulators are potential targets for therapeutics to NTM, especially in a T cell-suppressed population, and many ubiquitin ligases modulate pathogenesis and innate immunity during infections, including mycobacterial infections. Here, we investigated the role of an E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (CBLB), in immunocompromised mouse models of NTM infection. We found that CBLB is essential to prevent bacterial growth and dissemination. Cblb deficiency debilitated natural killer cells, inflammatory monocytes, and macrophages in vivo. However, Cblb deficiency in macrophages did not wane its ability to inhibit bacterial growth or production of reactive oxygen species or interferon γ production by natural killer cells in vitro. CBLB restricted NTM growth and dissemination by promoting early granuloma formation in vivo. Our study shows that CBLB bolsters innate immune responses and helps prevent the dissemination of NTM during compromised T cell immunity.

Decreased Interleukin-1 Family Cytokine Production in Patients with Nontuberculous Mycobacterial Lung Disease.

Nontuberculous mycobacteria (NTM) cause pulmonary disease in individuals without obvious immunodeficiency. This study was initiated to gain insight into the immunological factors that predispose persons to NTM pulmonary disease (NTMPD). Blood was obtained from 15 pairs of NTMPD patients and their healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the Mycobacterium avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in plasma and PBMC culture supernatants using multiplex immunoassays, and gene expression in the PBMCs was determined using real-time PCR. PBMCs from NTMPD patients produced significantly less interleukin-1ß (IL-1ß), IL-18, IL-1α, and IL-10 than PBMCs from their healthy household contacts in response to MAC. Although plasma RANTES levels were high in NTMPD patients, they had no effect on IL-1ß production by macrophages infected with MAC. Toll-like receptor 2 (TLR2) and TWIK2 (a two-pore domain K+ channel) were impaired in response to MAC in PBMCs of NTMPD patients. A TLR2 inhibitor decreased all four cytokines, whereas a two-pore domain K+ channel inhibitor decreased the production of IL-1ß, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. A reduced monocyte ratio might contribute to the attenuated production of IL-1 family cytokines by PBMCs of NTMPD patients in response to MAC stimulations. Collectively, our findings suggest that the attenuated IL-1 response may increase susceptibility to NTM pulmonary infection through multiple factors, including impaired expression of the TLR2 and TWIK2 and reduced monocyte ratio. IMPORTANCE Upon MAC stimulation, the production of IL-1 family cytokines and IL-10 by PBMCs of NTMPD patients was attenuated compared with that of healthy household contacts. Upon MAC stimulation, the expression of TLR2 and TWIK2 (one of the two-pore domain K+ channels) was attenuated in PBMCs of NTMPD patients compared with that of healthy household contacts. The production of IL-1 family cytokines by MAC-stimulated PBMCs and MAC-infected monocytes of healthy donors was reduced by a TLR2 inhibitor and two-pore domain K+ channel inhibitor. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. Collectively, our data suggest that defects in the expression of TLR2 and TWIK2 in human PBMCs or monocytes and reduced monocyte ratio are involved in the reduced production of IL-1 family cytokines, and it may increase susceptibility to NTM pulmonary infection.

Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection.

Mycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and ß-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited. IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

The Trend of TIM3 Expression on T Cells in Patients With Nontuberculous Mycobacterial Lung Disease: From Immune Cell Dysfunction to Clinical Severity.

Background: The incidence of nontuberculous mycobacterial lung disease (NTM-LD) is increasing worldwide. Immune exhaustion has been reported in NTM-LD, but T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a co-inhibitory receptor on T cells, has been scarcely studied. Methods: Patients with NTM-LD and healthy controls were prospectively recruited from July 2014 to August 2019 at three tertiary referral centers in Taiwan. We examined TIM3 expression on the T cells from the participants using flow cytometry. TIM3 expression was analyzed for different disease statuses and after treatment. The apoptosis and cytokine profiles were analyzed according to the TIM3 expression. Results: Among enrolled subjects (47 patients and 46 controls), TIM3 on CD4+ cells (6.44% vs. 4.12%, p = 0.028) and CD8+ cells (18.47% vs. 9.13%, p = 0.003) were higher in NTM-LD patients than in the controls. The TIM3 level on CD4+ and CD8+ T cells was positively associated with T-cell apoptosis in the NTM-LD patients. In stimulating peripheral blood mononuclear cells using PMA plus ionomycin, a high TIM3 level on T cells correlated with low interleukin-2 and tumor necrosis factor-alpha (TNF-α) on CD4+ cells and interferon-gamma and TNF-α on CD8+ T cells. For clinical manifestation, low body mass index (BMI), positive sputum acid-fast smear, and high radiographic score correlated with high TIM3 expression on T cells. After NTM treatment, TIM3+ decreased significantly on CD4+ and CD8+ T cells. Conclusions: In patients with NTM-LD, TIM3+ expression increased over CD4+ and CD8+ T cells and correlated with cell apoptosis and specific cytokine attenuation. Clinically, TIM3+ T cells increased in patients with low BMI, high disease extent, and high bacilli burden but decreased after treatment.

The role of interferon-gamma and interferon-gamma receptor in tuberculosis and nontuberculous mycobacterial infections.

Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM) remain the leading causes of lung disease and mortality worldwide. Interferon-gamma (IFN-γ) and its receptor (IFN-γR) play a key role in mediating immunity against Mtb and NTM. This study was conducted as a systematic review; all information was collected from databases such as: PubMed, Scopus, Medline, SID, and medical databases. Finally, all the collected data were reviewed, and all content was categorized briefly. There is growing evidence that IFN-γ plays an important role in host defense against these two intracellular pathogens by activating macrophages. In addition, IFN-γ has been shown to be an integral part of various antibacterial methods such as granuloma formation and phagosome-lysosome fusion, both of which lead to the death of intracellular Mycobacterium. As a result, its absence is associated with overgrowth of intracellular pathogens and disease caused by Mtb or Mycobacterium nontuberculosis. We also look at the role of IFN-γR in Mtb or NTM because IFN-γ acts through IFN-γR. Finally, we introduce new approaches to the treatment of M. tuberculosis complex (MTC) and NTM disease, such as cell and gene-based therapies that work by modulating IFN-γ and IFN-γR.

Type I Interferons Are Involved in the Intracellular Growth Control of Mycobacterium abscessus by Mediating NOD2-Induced Production of Nitric Oxide in Macrophages.

Mycobacterium abscessus (MAB) is one of the rapidly growing, multidrug-resistant non-tuberculous mycobacteria (NTM) causing various diseases including pulmonary disorder. Although it has been known that type I interferons (IFNs) contribute to host defense against bacterial infections, the role of type I IFNs against MAB infection is still unclear. In the present study, we show that rIFN-ß treatment reduced the intracellular growth of MAB in macrophages. Deficiency of IFN-α/ß receptor (IFNAR) led to the reduction of nitric oxide (NO) production in MAB-infected macrophages. Consistently, rIFN-ß treatment enhanced the expression of iNOS gene and protein, and NO production in response to MAB. We also found that NO is essential for the intracellular growth control of MAB within macrophages in an inhibitor assay using iNOS-deficient cells. In addition, pretreatment of rIFN-ß before MAB infection in mice increased production of NO in the lungs at day 1 after infection and promoted the bacterial clearance at day 5. However, when alveolar macrophages were depleted by treatment of clodronate liposome, rIFN-ß did not promote the bacterial clearance in the lungs. Moreover, we found that a cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2) is required for MAB-induced TANK binding kinase 1 (TBK1) phosphorylation and IFN-ß gene expression in macrophages. Finally, increase in the bacterial loads caused by reduction of NO levels was reversed by rIFN-ß treatment in the lungs of NOD2-deficient mice. Collectively, our findings suggest that type I IFNs act as an intermediator of NOD2-induced NO production in macrophages and thus contribute to host defense against MAB infection.

A Bumpy Ride of Mycobacterial Phagosome Maturation: Roleplay of Coronin1 Through Cofilin1 and cAMP.

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.

Autophagy and Host Defense in Nontuberculous Mycobacterial Infection.

Autophagy is critically involved in host defense pathways through targeting and elimination of numerous pathogens via autophagic machinery. Nontuberculous mycobacteria (NTMs) are ubiquitous microbes, have become increasingly prevalent, and are emerging as clinically important strains due to drug-resistant issues. Compared to Mycobacterium tuberculosis (Mtb), the causal pathogen for human tuberculosis, the roles of autophagy remain largely uncharacterized in the context of a variety of NTM infections. Compelling evidence suggests that host autophagy activation plays an essential role in the enhancement of antimicrobial immune responses and controlling pathological inflammation against various NTM infections. As similar to Mtb, it is believed that NTM bacteria evolve multiple strategies to manipulate and hijack host autophagy pathways. Despite this, we are just beginning to understand the molecular mechanisms underlying the crosstalk between pathogen and the host autophagy system in a battle with NTM bacteria. In this review, we will explore the function of autophagy, which is involved in shaping host-pathogen interaction and disease outcomes during NTM infections. These efforts will lead to the development of autophagy-based host-directed therapeutics against NTM infection.

The Role of Galanin during Bacterial Infection in Larval Zebrafish.

Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.

Integrative Analysis of Human Macrophage Inflammatory Response Related to Mycobacterium tuberculosis Virulence.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, kills 1.5 to 1.7 million people every year. Macrophages are Mtb's main host cells and their inflammatory response is an essential component of the host defense against Mtb. However, Mtb is able to circumvent the macrophages' defenses by triggering an inappropriate inflammatory response. The ability of Mtb to hinder phagolysosome maturation and acidification, and to escape the phagosome into the cytosol, is closely linked to its virulence. The modulation of the host inflammatory response relies on Mtb virulence factors, but remains poorly studied. Understanding macrophage interactions with Mtb is crucial to develop strategies to control tuberculosis. The present study aims to determine the inflammatory response transcriptome and miRNome of human macrophages infected with the virulent H37Rv Mtb strain, to identify macrophage genetic networks specifically modulated by Mtb virulence. Using human macrophages infected with two different live strains of mycobacteria (live or heat-inactivated Mtb H37Rv and M. marinum), we quantified and analyzed 184 inflammatory mRNAs and 765 micro(mi)RNAs. Transcripts and miRNAs differently modulated by H37Rv in comparison with the two other conditions were analyzed using in silico approaches. We identified 30 host inflammatory response genes and 37 miRNAs specific for H37Rv virulence, and highlight evidence suggesting that Mtb intracellular-linked virulence depends on the inhibition of IL-1ß-dependent pro-inflammatory response, the repression of apoptosis and the delay of the recruitment and activation of adaptive immune cells. Our findings provide new potential targets for the development of macrophage-based therapeutic strategies against TB.

Potent antibody-mediated neutralization limits bacteriophage treatment of a pulmonary Mycobacterium abscessus infection.

An 81-year-old immunocompetent patient with bronchiectasis and refractory Mycobacterium abscessus lung disease was treated for 6 months with a three-phage cocktail active against the strain. In this case study of phage to lower infectious burden, intravenous administration was safe and reduced the M. abscessus sputum load tenfold within one month. However, after two months, M. abscessus counts increased as the patient mounted a robust IgM- and IgG-mediated neutralizing antibody response to the phages, which was associated with limited therapeutic efficacy.