Satoru Mizuno

Mycobacterial infection in TLR2 and TLR6 knockout mice.

To investigate the role of TLR in the development of murine tuberculosis in vivo, TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild‐type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF‐α, TGF‐β, IL‐1β, and IL‐2 were significantly lower, but levels of the mRNAs for IL‐4 and IL‐6 were higher, than in wild‐type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF‐κB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.

Role of Interleukin (IL)-1 Type 1 Receptor in Mycobacterial Infection

It is important to gain a better understanding of IL‐1‐mediated signaling events in mycobacterial infection. In order to clarify the role of IL‐1 receptor type 1 (IL‐1 R1) in IL‐1 R1, knockout (KO) mice were infected with either Mycobacterium tuberculosis H37Rv or Kurono strain by the respiratory route, and their ability to control mycobacterial growth, pulmonary granuloma formation, and cytokine mRNA expression was investigated. IL‐1 R1 KO mice developed significantly larger (P < 0.01) granulomatous lesions with neutrophil infiltration in their lungs than wild‐type mice did after infection with the M. tuberculosis Kurono strain. The number of mycobacterial colonies in lungs and spleen increased from five weeks post‐infection. Interferon‐γ production by spleen cells was low in IL‐1 R1 KO mice. It is concluded that the IL‐1 R1 is essential for IL‐1‐mediated signaling events in mycobacterial infection.

IL-4 is required for defense against mycobacterial infection

Although the involvement of T helper (Th1) cells is central to protection against intracellular bacteria, including Mycobacterium tuberculosis, the involvement of Th2 cells, characterized by potent interleukin (IL)‐4 secretion in mycobacterial infection is still unclear. In order to clarify the role of IL‐4 in murine tuberculosis, IL‐4‐deficient mutant mice, IL‐4 knockout (IL‐4 KO) mice, were utilized. The mice were infected with H37Rv, Kurono or BCG Pasteur via an airborne infection route by placing them in the exposure chamber of a Middlebrook airborne infection apparatus. Their capacity to control mycobacterial growth, granuloma formation, cytokine secretion, and nitric oxide (NO) production were examined. These mice developed large granulomas, but not necrotic lesions in the lungs, liver or spleen (P < 0.05). This was consistent with a significant increase in lung colony‐forming units (CFU). Compared with levels in wild‐type mice, upon stimulation with mycobacteria, splenic IL‐10 levels were low and IL‐6 levels were intermediate, but interferon (IFN)‐γ and IL‐12 levels were significantly higher. IL‐18 levels were within the normal range. The level of NO production by alveolar macrophages of the IL‐4 KO mice was similar to that of the wild‐type mice. Granulomatous lesion development by IL‐4 KO mice was inhibited significantly by treatment with exogenous recombinant IL‐4. These findings were not specific to the IL‐4 KO mice used. Our data show that IL‐4 may play a protective role in defense against mycobacteria, although IFN‐γ and TNF‐α play major roles in it. Our data do not rule out an IFN‐γ‐independent function of IL‐4 in controlling tuberculosis.

Relative Importance of NF-κB p50 in Mycobacterial Infection

ABSTRACT To understand the role of NF-κB in the development of murine tuberculosis in vivo, NF-κB p50 knockout mice were infected withMycobacterium tuberculosis by placing them in the exposure chamber of an airborne-infection apparatus. These mice developed multifocal necrotic pulmonary lesions or lobar pneumonia. Compared with the levels in wild-type mice, pulmonary inducible nitric oxide synthase, interleukin-2 (IL-2), gamma interferon, and tumor necrosis factor alpha mRNA levels were significantly low but expression of IL-10 and transforming growth factor β mRNAs were within the normal ranges. The pulmonary IL-6 mRNA expression level was higher. Therefore, NF-κB and its interaction with host cells play an important role in the pathogenesis of tuberculosis.

Interleukin-15 as an immune adjuvant to increase the efficacy of Mycobacterium bovis bacillus Calmette-Guérin vaccination.

ABSTRACT Interleukin-15 (IL-15) transgenic mice which had been inoculated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) 24 weeks previously showed resistance against airborne infection with Mycobacterium tuberculosis H37Rv accompanied by an increased CD8+-Tc1-cell response. IL-15 may be used as an immune adjuvant given with BCG vaccination to enhance its biologic efficacy.

Disruption of Nuclear Factor-Interleukin-6, a Transcription Factor, Results in Severe Mycobacterial Infection

Nuclear factor-interleukin-6 (NF-IL-6) is one of several nuclear transcription factors (NF-IL-6, NF-kappaB, PU.1, interferon-regulatory factor 1, Egr-1, and Stat-1). NF-IL-6 and NF-kappaB are expressed in macrophages and is induced by bacterial lipopolysaccharides. To evaluate whether NF-IL-6 is required for the inflammatory immune response to mycobacterial infection, in which epithelioid macrophages comprise the leading cell population, we generated NF-IL-6 knockout (KO) mutant mice. Airborne infection of these mice with Mycobacterium tuberculosis strains induced disseminated tuberculosis lacking granuloma formation, although interferon-gamma, tumor necrosis factor-alpha, and interleukin-12 mRNA expression levels were within the normal range compared with those of wild-type mice. Generation of O2- and mycobacterial killing by neutrophils from these mice were impaired severely compared with wild-type mice. We conclude that NF-IL-6 is a critical transcription factor in mycobacterial control as well as in granulocyte-colony stimulating factor induction resulting in neutrophil activation.

Interferon regulatory factor 1 in mycobacterial infection

In order to understand the role of IRF‐1 in the development of murine tuberculosis in vivo, IRF‐1 knockout mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. These knockout mice developed multifocal necrotic lesions in the lung, liver and spleen tissues and died of disseminated tuberculosis within 43 days of infection. Compared with the levels in wild‐type mice, the pulmonary inducible NO synthase (iNOS) mRNA expression level was significantly lower, but IL‐18 and IL‐6 mRNA levels were higher. There was no statistically significant difference in the expression of IFN‐γ and TNF‐α mRNA between the IRF‐1 knockout and wild‐type mice. IRF‐1 is indirectly responsible for iNOS mRNA expression and plays an important role in the pathogenesis of murine tuberculosis.

Pathological and immunological profiles of rat tuberculosis

To investigate the pathological and immunological profiles of rat tuberculosis, Lewis female rats were infected aerially with Mycobacterium tuberculosis. Histopathology, immunological profiles of mononuclear cells from M. tuberculosis‐infected rat lung tissue, and the expression patterns of cytokine and iNOS mRNAs were examined over time. M. tuberculosis induced granulomatous lesions in the lungs, spleen, lymph nodes and liver, but these lesions lacked central necrosis. Multinucleate giant cells were observed in late‐phase tuberculosis. CD4+ and CD8+ T cells increased with time and reached a peak 5 weeks after infection, decreasing gradually thereafter. ED1 antigen, suggestive of alveolar macrophages, was expressed at a high level in early phase tuberculosis and remained at the same level even in the late phase. OX62 antigen increased gradually and reached a peak 5 weeks after infection. Interferon‐γ, tumour necrosis factor‐α and iNOS mRNAs were expressed strongly over time, but their expression decreased 12 weeks after infection. Because rat tuberculosis is very similar to murine tuberculosis and it is easy to obtain mononuclear cells from M. tuberculosis‐infected rat lung tissue, the rat tuberculosis model appears to be suitable for immunological studies in vivo.

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.

Nude rat (F344/N-rnu) tuberculosis

As many mononuclear cells from Mycobacterium tuberculosis‐infected lung tissues are not available for fluorescence‐activated cell sorter (FACS) analysis and the tuberculin test is not feasible in a mouse tuberculosis model, we attempted to develop a rat tuberculosis model. We have previously reported that rat tuberculosis is associated with granulomas that lack central necrosis. In order to develop a better animal model of tuberculosis in immunocompromised humans (tuberculosis associated with HIV infection or tuberculosis of the elderly), we infected F344/N‐rnu nude rats with M. tuberculosis via the airborne route. The animals developed pulmonary granulomas with central necrosis encapsulated by dense collagen fibres, closely resembling those of human tuberculosis. The nude rats died of disseminated tuberculosis by the 85th day after aerosol infection, while F344 wild‐type rats did not. Interestingly, T‐cells that were reactive with anti‐CD4 antibody and anti‐CD8 antibody, indicating the presence of remnant thymus, were observed in the infected lung tissues of the nude rats. Therefore, T‐cell precursors may be present in nude rats. The nude rat tuberculosis model mimics tuberculosis in immunocompromised humans and may provide a suitable model for immunological studies in vivo.