Mucosal-associated invariant T (MAIT) cells play an important physiological role in host pathogen defense and may also be involved in inflammatory disorders and multiple sclerosis. The rarity and inefficient expansion of these cells have hampered detailed analysis and application. Here, we report an induced pluripotent stem cell (iPSC)-based reprogramming approach for the expansion of functional MAIT cells. We found that human MAIT cells can be reprogrammed into iPSCs using a Sendai virus harboring standard reprogramming factors. Under T cell-permissive conditions, these iPSCs efficiently redifferentiate into MAIT-like lymphocytes expressing the T cell receptor Vα7.2, CD161, and interleukin-18 receptor chain α. Upon incubation with bacteria-fed monocytes, the derived MAIT cells show enhanced production of a broad range of cytokines. Following adoptive transfer into immunocompromised mice, these cells migrate to the bone marrow, liver, spleen, and intestine and protect against Mycobacterium abscessus. Our findings pave the way for further functional analysis of MAIT cells and determination of their therapeutic potential.

BackgroundEpidemiological studies have suggested that suspended particulate matter (SPM) causes detrimental health effects such as respiratory and cardiovascular diseases, and that diesel exhaust particles from automobiles is a major contributor to SPM. It has been reported that neonatal and adult exposure to diesel exhaust damages the central nervous system (CNS) and induces behavioral alteration. Recently, we have focused on the effects of prenatal exposure to diesel exhaust on the CNS. In this study, we examined the effects of prenatal exposure to low concentration of diesel exhaust on behaviour and the monoaminergic neuron system. Spontaneous locomotor activity (SLA) and monoamine levels in the CNS were assessed.MethodsMice were exposed prenatally to a low concentration of diesel exhaust (171 μg DEP/m3) for 8 hours/day on gestational days 2-16. SLA was assessed for 3 days in 4-week-old mice by analysis of the release of temperature-associated infrared rays. At 5 weeks of age, the mice were sacrificed and the brains were used for analysis by high-performance liquid chromatography (HPLC).Results and DiscussionMice exposed to a low concentration of diesel exhaust showed decreased SLA in the first 60 minutes of exposure. Over the entire test period, the mice exposed prenatally to diesel exhaust showed decreased daily SLA compared to that in control mice, and the SLA in each 3 hour period was decreased when the lights were turned on. Neurotransmitter levels, including dopamine and noradrenaline, were increased in the prefrontal cortex (PFC) in the exposure group compared to the control group. The metabolites of dopamine and noradrenaline also increased in the PFC. Neurotransmitter turnover, an index of neuronal activity, of dopamine and noradrenaline was decreased in various regions of the CNS, including the striatum, in the exposure group. The serum corticosterone level was not different between groups. The data suggest that decreased SLA in mice exposed prenatally to diesel exhaust is due to facilitated release of dopamine in the PFC.ConclusionsThese results indicate that exposure of mice in utero to a low concentration of diesel exhaust decreases SLA and alters the neurochemical monoamine metabolism of several regions of the brain.

To understand the role of neutrophils in the development of rat tuberculosis in vivo, we utilized lipopolysaccharide (LPS)-induced neutrophilia in the lungs. LPS (50 g/ml) was administered intratracheally to male Fischer rats. Rats were then infected with Mycobacterium tuberculosis by an airborne route. Intratracheal injection of LPS significantly blocked the development of pulmonary granulomas and significantly reduced pulmonary CFU (P < 0.01). LPS treatment with amphotericin B (an LPS inhibitor) or neutralizing anti-rat neutrophil antibody reversed the development of pulmonary lesions. LPS-induced transient neutrophilia prevented early mycobacterial infection. The timing of LPS administration was important. When given intratracheally at least 10 days after aerial infection, LPS did not prevent development of tuberculosis. Neutrophils obtained by bronchoalveolar lavage killed M. tuberculosis cells. These results indicate clearly that neutrophils participate actively in defense against early-phase tuberculosis.When tubercle bacilli are introduced into the lung, neutrophils migrate and accumulate in the infected pulmonary lesions during the early stage of tuberculosis. This rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis (5). However, it has been reported that murine neutrophils play a protective nonphagocytic role in systemic M. tuberculosis infection in mice (3). Neutrophils from M. avium-infected mice produce tumor necrosis factor alpha, interleukin-12 (IL-12), and IL-1␤ (4). Thus, little is known about immunologic function in the lungs (although neutrophils have been identified as sources of inflammatory cytokines and chemokines) (2). In circumstances in which neutrophils are not activated with granulocyte colonystimulating factor, severe mycobacterial infection results (7). However, it remains unclear whether neutrophils play a protective role in defense against mycobacterial infection. In vivo studies of neutrophils in the lungs are challenging, because it is difficult to induce neutrophilia. We therefore modified the lipopolysaccharide (LPS)-induced neutrophilia system in rats so that we could utilize it to study rat tuberculosis (1).Permission to experiment on animals was given by the Animal Experiment Committee of The Research Institute of Tuberculosis. We injected LPS (E. coli 0111:B4 LPS) purchased from Sigma (St. Louis, Mo.) (50 g/0.5 ml) intratracheally into anesthetized 6-week-old female Fischer rats (three rats/group). On the day after the injection, the rats were infected with the M. tuberculosis Kurono strain (ATCC 25618) by an airborne route by placing them in the exposure chamber of an airborne infection apparatus (model 099CA4241; Gals-Col, Inc., Terre Haute, Ind.). The concentration was calculated to result in the uptake of around 200 viable bacilli by the rat lungs after inhalation exposure for 90 min under the experimental conditions of this study (8). For the pulmonary CFU assay, 7 weeks later the lungs were rem...

To gain a better understanding of the pathological role of interferon-y (IFN-y) in specific granuloma formation, IFN-y gene-deficient mice (BALB/c and C57BL/6) were produced. The IFN-y gene in embryonic stem (ES) cells was disrupted by inserting the P-galactosidase gene (IacZ) and the neomycin resistance gene ( m u ) at the translation initiation site in exon 1 by homologous recombination. Six-week-old IFN-y-deficient and wild-type mice were inoculated with lo3 -10' bacilli of various strains of Mycobacteriurn tuberculosis (Kurono, H37Rv, H37Ra and BCG Pasteur) through their tail veins. The mice were examined 7 weeks later for granuloma formation. The avirulent BCG Pasteur and H37Ra strains (lo3-lo4 bacilli/ml) induced granulomas in the spleen, liver and lungs of IFN-y-deficient mice. The granulomas consisted of epithelioid macrophages and Langhans multinucleate giant cells, but lacked caseous necrosis. The virulent Kurono and H37Rv strains induced disseminated abscesses but not granulomas in various organs of IFN-y-deficient mice and Mac-3-positive macrophages were not detected in the abscess lesions. These results suggest that IFN-y may be primarily responsible for macrophage activation and that other factor(s) may be involved in the granuloma formation mechanism.

No abstract

CD4(+)CD25(+) regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4(+)CD25(+) Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25(+) cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25(+) cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4(+)CD25(-) T cells alone or a combination of CD4(+)CD25(+) and CD4(+)CD25(-) T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4(+)CD25(+) Treg cells to CD4(+)CD25(-) effector T cells, as demonstrated by the lack of response of CD4(+)CD25(+) T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis. Our data show that CD4(+)CD25(+) Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection.

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.

Guinea pigs are often used as an animal model of human tuberculosis (TB