Yutaka Tatano

Characteristics of Suppressor Macrophages Induced by Mycobacterial and Protozoal Infections in relation to Alternatively Activated M2 Macrophages

In the advanced stages of mycobacterial infections, host immune systems tend to change from a Th1-type to Th2-type immune response, resulting in the abrogation of Th1 cell- and macrophage-mediated antimicrobial host protective immunity. Notably, this type of immune conversion is occasionally associated with the generation of certain types of suppressor macrophage populations. During the course of Mycobacterium tuberculosis (MTB) and Mycobacterium avium-intracellulare complex (MAC) infections, the generation of macrophages which possess strong suppressor activity against host T- and B-cell functions is frequently encountered. This paper describes the immunological properties of M1- and M2-type macrophages generated in tumor-bearing animals and those generated in hosts with certain microbial infections. In addition, this paper highlights the immunological and molecular biological characteristics of suppressor macrophages generated in hosts with mycobacterial infections, especially MAC infection.

Recent advances in antituberculous drug development and novel drug targets

TB, especially multidrug-resistant TB and extensively drug-resistant TB, is an important global health concern, and the novel development of effective anti-tuberculous drugs is urgently needed. Newly elucidated, critical information on the entire genome of Mycobacterium tuberculosis (MTB) and advances in knowledge regarding various mycobacterial virulence genes are promoting progression in the identification of genes that code for new drug targets. With this background, this review deals with the following areas: first, the future development of new anti-tuberculous drugs is discussed according to the potential pharmacological targets of MTB; and second, a review of the present development status of new anti-tuberculous drugs is conducted, particularly focusing on some promising new anti-tuberculous agents, such as nitroimidazoles, diarylquinolines and oxazolidinones.

Unique macrophages different from M1/M2 macrophages inhibit T cell mitogenesis while upregulating Th17 polarization.

Mycobacterial infection induces suppressor macrophages (MΦs), causing disease exacerbation. There are two major MΦ subsets (M1 and M2 MΦs) that are phenotypically and functionally different. Here, we examined which of the MΦ subsets the mycobacterial infection-induced suppressor MΦs (MIS-MΦs) belong to. MIS-MΦs down-regulated T cell production of Th1 and Th2 cytokines but markedly increased production of interleukin (IL)-17A and IL-22 through up-regulation of Th17 cell expansion. In this phenomenon, a novel MΦ population, which is functionally distinguishable from M1 and M2 MΦ subsets and possesses unique phenotypes (IL-12+, IL-1βhigh, IL-6+, tumor necrosis factor (TNF)-α+, nitric oxide synthase (NOS) 2+, CCR7high, IL-10high, arginase (Arg)-1−, mannose receptor (MR)low, Ym1high, Fizzlow, and CD163high), played central roles through the action of IL-6 and transforming growth factor (TGF)-β but not IL-21 and IL-23. This new type of MΦ population was induced in infected mice and actively supported the in vivo expansion of Th17 cells.

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

Development of new antituberculous drugs based on bacterial virulence factors interfering with host cytokine networks.

The worldwide increase in the prevalence of tuberculosis (TB), especially multidrug-resistant TB and extensively drug-resistant TB, is an important global health concern, and new effective drugs are urgently needed. Information on the genome of Mycobacterium tuberculosis (MTB) and various mycobacterial virulence genes is leading to the identification of genes that code for new drug targets. Mycobacterium tuberculosis (MTB) is resistant to the antimicrobial mechanisms of host macrophages and can survive and replicate in macrophages for long periods, resulting in a persistent infection. Mycobacterial virulence factors suppress macrophage bactericidal functions partly via their downregulatory effects on the host antimicrobial cytokine networks, consisting of proinflammatory, immunopotentiating, and Th1-inducing cytokines. Thus, for the development of unique drugs that exhibit antimycobacterial action through novel mechanisms, it is reasonable to search for targets among bacterial genes encoding virulence factors which interfere with the host cytokine responses protective to mycobacterial pathogens. In this review, we discuss the profiles of cytokine networks related to host resistance to mycobacteria, including the mechanisms of downregulation of host antimycobacterial immunity due to immunosuppressive cytokines, which are occasionally induced in the advanced stages of TB. We also highlight the development of antituberculous drugs based on bacterial virulence factors interfering with the host antimycobacterial cytokine network.

Comparative in vitro and in vivo antimicrobial activities of sitafloxacin, gatifloxacin and moxifloxacin against Mycobacterium avium

Moxifloxacin exhibits therapeutic activity against Mycobacterium avium infection in mice. Since not only moxifloxacin but also another 8-methoxy quinolone, gatifloxacin, and a C-8-chloro quinolone, sitafloxacin, show favourable antimycobacterial activity in vitro, their anti-M. avium activities were compared in vivo. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and mutant prevention concentrations (MPCs) of the test quinolones for M. avium were determined by microdilution in 7HSF broth. Antimicrobial activity against intracellular bacteria was measured using Mono Mac 6 human macrophages. Therapeutic efficacy of the quinolones when administered subcutaneously with or without clarithromycin plus ethambutol was assessed using mice intravenously infected with M. avium in terms of changes in bacterial loads in the lungs and spleen following infection. Based on the MICs, MBCs and MPCs, the in vitro activities of sitafloxacin and moxifloxacin were greater than that of gatifloxacin. Moxifloxacin exhibited the strongest activity against intramacrophage M. avium. When each test quinolone was administered alone to infected mice, sitafloxacin and gatifloxacin exhibited greater therapeutic efficacy than moxifloxacin based on intrapulmonary bacterial elimination. However, moxifloxacin exerted greater activity in killing bacteria in the spleen. Moxifloxacin and sitafloxacin exhibited combined effects on intrapulmonary bacterial elimination when administered to mice in combination with clarithromycin plus ethambutol. Sitafloxacin exerted the most marked combined effects in bacterial killing in the spleen. Levofloxacin displayed the lowest in vitro and in vivo activities amongst the tested quinolones. Taken together, these findings indicate that sitafloxacin and moxifloxacin exhibit favourable activities against M. avium in vitro and in vivo.

ATP Exhibits Antimicrobial Action by Inhibiting Bacterial Utilization of Ferric Ions

ATP up-regulates macrophage antimycobacterial activity in a P2X7-dependent manner, but little is known about whether ATP directly exhibits antimicrobial effects against intracellular mycobacteria. In this study, we found that ATP inhibited the growth of various bacteria, including Staphylococcus, Pseudomonas, and mycobacteria, without damaging bacterial surface structures. Using gene technology, we newly established an enterobactin-deficient (entB−) mutant from ATP-resistant Klebsiella pneumoniae, and found the recovery of ATP susceptibility in the enterobactin-deleted mutant. Therefore, ATPs antibacterial activity is attributable to its iron-chelating ability. Since ATP distributed in the cytosol of macrophages at high concentrations, ATP appears to augment macrophages antimicrobial activity by directly attacking intracytosolic and intra-autophagosomal pathogens. Furthermore, ATP exhibited combined effects with some antimicrobials against methicillin-resistant S. aureus (MRSA) and M. intracellulare, suggesting its usefulness as an adjunctive drug in the chemotherapy of certain intractable infections.

Correlation between variable-number tandem-repeat-based genotypes and drug susceptibility in Mycobacterium avium isolates

Little is known about the correlation between genotype and drug susceptibility in Mycobacterium avium (Mav) strains isolated from patients with Mav infections. To examine whether drug susceptibility profile of Mav is associated with genotype, we carried out variable-number tandem-repeat (VNTR) typing and drug susceptibility testing for Mav isolates from Japanese with nodular-bronchiectasis (NB)-type and cavitary disease (CA)-type diseases. We performed M. avium tandem repeat (MATR)-VNTR typing and drug susceptibility testing by the broth dilution method, using macrolides, rifamycins, ethambutol, isoniazid, aminoglycosides, and quinolones, for Mav isolates from patients with NB and CA-type diseases (NB-Mav and CA-Mav). Based on the VNTR genotyping, the Mav strains were grouped into three clusters. There was no difference with respect to the distribution of NB-Mav and CA-Mav among the clusters. We observed a strong association between VNTR genotype and susceptibility to quinolones (levofloxacin, moxifloxacin, gatifloxacin, sitafloxacin, and garenoxacin) and ethambutol. There was essentially no significant difference in drug susceptibility between NB- and CA-Mav strains, although NB-Mav was somewhat more resistant to fluoroquinolones, especially gatifloxacin, than CA-Mav. There was a significant association between VNTR genotype and susceptibility to quinolones and ethambutol in Mav isolates from Japanese patients.

Comparative study for the virulence of Mycobacterium avium isolates from patients with nodular-bronchiectasis- and cavitary-type diseases

Mycobacterium avium (Mav) lung infections, called nodular-bronchiectasis (NB)-type M. avium complex (MAC) disease, are globally increasing. To elucidate whether there are unusual populations of Mav, causing NB-type disease rather than cavitary (CA)-type disease, we compared the virulence of Mav isolates from patients with NB-type (NB-Mav) and those from CA-type (CA-Mav) diseases, based on intracellular growth in various types of human cells. Five strains each of NB-Mav and CA-Mav were compared with each other for their invasiveness and ability to intracellularly replicate in various types of cultured cells of human origin. The two types of Mav isolates showed a similar ability, on average, to replicate in macrophages and lung epithelial cells. Moreover, they showed a similar ability to induce the production of reactive nitrogen intermediates and reactive oxygen intermediates by macrophages and susceptibility to antimicrobial molecules. Therefore, it appears that there is no essential difference in virulence in terms of infectivity to human macrophages and lung cells between Mav strains isolated from NB-MAC disease and those from CA-MAC disease. These findings indicate the importance of further studies to elucidate the mechanism for the establishment of NB-type MAC diseases based on host immunological conditions rather than the pathogenic nature of MAC organisms themselves.

Aldose reductase participates in the downregulation of T cell functions due to suppressor macrophages.

The cell-to-cell contact of T lymphocytes with immunosuppressive macrophages causes marked changes in the tyrosine phosphorylation of some cytosolic proteins of T cells. By phosphoproteome analysis, we identified a 36-kDa protein as aldose reductase (AR). The AR expression in T cells was not changed by TCR stimulation or due to cell-to-cell transmission of suppressor signals from immunosuppressive macrophages. Therefore, AR phosphorylation/dephosphorylation is essential for the transduction of TCR-mediated T-cell stimulatory signals, and moreover plays important roles for the cross-talk of immunosuppressive macrophage-derived suppressor signals with the signaling pathways for T-cell activation. Moreover, AR played important roles in the upregulation of ERK1/2-mediated signaling pathways in T lymphocytes. Notably, the enzymatic activity of AR was not required for its signaling action. Taken together, it is concluded that AR mediates intracellular transmission of the suppressor signal of immunosuppressive macrophages toward downstream ERK1/2 pathways, possibly through its direct interaction with acceptor proteins.