Satomi Yara

Monocyte Chemoattractant Protein-1-Dependent Increase of Vα14 NKT Cells in Lungs and Their Roles in Th1 Response and Host Defense in Cryptococcal Infection

To elucidate the role of NKT cells in the host defense to cryptococcal infection, we examined the proportion of these cells, identified by the expression of CD3 and NK1.1, in lungs after intratracheal infection with Cryptococcus neoformans. This population increased on day 3 after infection, reached a peak level on days 6–7, and decreased thereafter. In Vα14 NKT cell-deficient mice, such increase was significantly attenuated. The proportion of Vα14 NKT cells, detected by binding to α-galactosylceramide-loaded CD1d tetramer, and the expression of Vα14 mRNA increased after infection with a similar kinetics. The delayed-type hypersensitivity response and differentiation of the fungus-specific Th1 cells was reduced in Vα14 NKT cell-deficient mice, compared with control mice. Additionally, elimination of this fungal pathogen from lungs was significantly delayed in Vα14 NKT cell-deficient mice. Production of monocyte chemoattractant protein (MCP)-1 in lungs, detected at both mRNA and protein levels, increased on day 1, reached a peak level on day 3, and decreased thereafter, which preceded the increase in NKT cells. Finally, the increase of total and Vα14+ subset of NKT cells after infection was significantly reduced in MCP-1-deficient mice. Our results demonstrated that NKT cells, especially Vα14+ subset, accumulated in a MCP-1-dependent manner in the lungs after infection with C. neoformans and played an important role in the development of Th1 response and host resistance to this fungal pathogen.

Activation of Vα14+ Natural Killer T Cells by α-Galactosylceramide Results in Development of Th1 Response and Local Host Resistance in Mice Infected with Cryptococcus neoformans

ABSTRACT We examined the effect of α-galactosylceramide (α-GalCer) on the synthesis of gamma interferon (IFN-γ) and local resistance in mice infected intravenously with Cryptococcus neoformans. The level of IFN-γ in serum increased on day 3, reached a peak level on day 7, and decreased to the basal level on day 14 postinfection in mice treated with α-GalCer, while in vehicle-treated mice, no increase was detected at any time points except for a small increase on day 7. Such effects were not observed in NKT-KO mice. In CD4KO mice, minor synthesis of IFN-γ was detected on day 3 in sera but was completely abolished by day 7. The α-GalCer-induced IFN-γ production on day 3 was partially reduced in mice depleted of NK cells by treatment with anti-asialo-GM1 antibody (Ab). Spleen cells obtained from infected and α-GalCer-treated mice on day 7 produced a large amount of IFN-γ upon restimulation with live organisms, while only a marginal level of production was detected in splenocytes from infected and vehicle-treated mice. Such effects were abolished in CD4KO and NKT-KO mice. Finally, the fungal loads in the lungs and spleen on days 7 and 14 were significantly reduced in α-GalCer-treated mice compared to those in control mice. In NKT-KO mice, local resistance elicited by α-GalCer was completely abolished, although no obvious exacerbation of infection was detected. Furthermore, treatment with anti-IFN-γ monoclonal Ab mostly abrogated the protective effect of this agent. Thus, our results indicated that activation of Vα14+ NKT cells resulted in an increased Th1 response and local resistance to C. neoformans through production of IFN-γ.

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-γ production by NK cells.

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40−/− mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-γ was still detected in these mice at a considerable level (20–30% of that in control mice). The host resistance was moderately impaired in IL-12p40−/− mice compared with IFN-γ−/− mice. Neutralizing anti-IFN-γ mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-γ and also impaired the host resistance. Host resistance in IL-12p40−/− IL-18−/− mice was more profoundly impaired than in IL-12p40−/− mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-γ and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40−/− mice did not produce any IFN-γ upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-γ. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-γ in IL-12p40−/− mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-γ production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.

Contribution of IL-18 to Th1 Response and Host Defense Against Infection by Mycobacterium tuberculosis: A Comparative Study with IL-12p40

The present study was conducted to critically determine the protective role of IL-18 in host response to Mycobacterium tuberculosis infection. IL-18-deficient (knockout (KO)) mice were slightly more prone to this infection than wild-type (WT) mice. Sensitivity of IL-12p40KO mice was lower than that of IL-12p40/IL-18 double KO mice. IFN-γ production caused by the infection was significantly attenuated in IL-18KO mice compared with WT mice, as indicated by reduction in the levels of this cytokine in sera, spleen, lung, and liver, and its synthesis by spleen cells restimulated with purified protein derivatives. Serum IL-12p40 level postinfection and its production by peritoneal exudate cells stimulated with live bacilli were also significantly lower in IL-18KO mice than WT mice, suggesting that attenuated production of IFN-γ was secondary to reduction of IL-12 synthesis. However, this was not likely the case, because administration of excess IL-12 did not restore the reduced IFN-γ production in IL-18KO mice. In further studies, IL-18 transgenic mice were more resistant to the infection than control littermate mice, and serum IFN-γ level and its production by restimulated spleen cells were increased in the former mice. Taken together, our results indicate that IL-18 plays an important role in Th1 response and host defense against M. tuberculosis infection although the contribution was not as profound as that of IL-12p40.

Enhanced Gamma Interferon Production through Activation of Vα14 + Natural Killer T Cells by α-Galactosylceramide in Interleukin-18-Deficient Mice with Systemic Cryptococcosis

ABSTRACT We showed recently that activation of Vα14+ natural killer T cells (NKT cells) by α-galactosylceramide (α-GalCer) resulted in increased gamma interferon (IFN-γ) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to α-GalCer-induced IFN-γ synthesis. Here we examined the role of IL-18 in α-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-γ in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-γ correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-γ synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-γ in WT mice. Finally, the enhanced production of IFN-γ in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in α-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-γ synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Vα14+ NKT cells.

NK Cells Eliminate Cryptococcus neoformans by Potentiating the Fungicidal Activity of Macrophages Rather than by Directly Killing Them upon Stimulation with IL‐12 and IL‐18

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)‐12 and IL‐18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN‐γ and exerted marked cytotoxic activity against YAC‐1 cells. On the other hand, NK cells significantly potentiated the nitric oxide‐mediated cryptococcocidal activity of thioglycolate‐elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL‐12 and IL‐18. The culture supernatants of NK cells stimulated with IL‐12 and IL‐18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN‐γ because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage‐mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.

Minimal Contribution of Vα14 Natural Killer T Cells to Th1 Response and Host Resistance against Mycobacterial Infection in Mice

We elucidated the contribution of Vα14 NKT cells to Th1 response and host resistance against mycobacterial infection. In Vα14 NKT cell‐deficient mice, host defense and DTH response to Mycobacterium bovis BCG were not different from wild‐type mice after pulmonary infection. There was no significant difference in the lung concentrations of IFN‐γ between the two strains of mice. In addition, host defense to systemic infection with M. tuberculosis was similar to that of M. bovis. Our results indicate that Vα14 NKT cells play only a marginal role, if any, in the Th1 response and host resistance to mycobacterial infection.

CT Scans of the Chest in Carriers of Human T-cell Lymphotropic Virus Type 1 : Presence of Interstitial Pneumonia

RATIONALE AND OBJECTIVES To evaluate pulmonary findings on computed tomography (CT) scans in carriers of human T-lymphotropic virus type 1 (HTLV-1). MATERIALS AND METHODS This retrospective study was approved by the Institutional Review Board at each institution, and informed consent was waived. Patients who were diagnosed with adult T-cell lymphoma/leukemia or collagen vascular disease were excluded from the study. Chest CT of 106 HTLV-1 carriers (54 females and 52 males; age range 44-94 years) were initially evaluated by two chest radiologists. Assessed CT findings included centrilobular nodules, thickening of bronchovascular bundles, ground-glass opacity, bronchiectasis, interlobular septal thickening, consolidation, honeycombing, crazy-paving appearance, enlarged lymph nodes, pleural effusion, and pericardial effusion. Three chest radiologists secondarily evaluated the CT scans with the abnormal findings to judge the presence of interstitial pneumonia patterns or a bronchiolitis/bronchitis pattern. RESULTS Abnormal CT findings were found in 65 (61.3%) patients, including ground-glass opacity (n = 33), bronchiectasis (n = 28), centrilobular nodules (n = 25), and interlobular septal thickening (n = 19). Honeycombing (n = 5) and crazy-paving appearance (n = 3) were also observed. Based on the CT findings, 10 subjects were diagnosed with interstitial pneumonia (usual interstitial pneumonia pattern, n = 3; nonspecific interstitial pneumonia pattern, n = 5; organizing pneumonia pattern, n = 2; respectively). Twenty subjects were diagnosed with the bronchitis/bronchiolitis pattern. CONCLUSION Although the bronchiolitis/bronchitis pattern is predominant on chest CT in HTLV-1 carriers, the HTLV-1 infection is associated with various interstitial pneumonias.

Computed tomographic features of 23 sporadic cases with Legionella pneumophila pneumonia

OBJECTIVE To describe the chest computed tomographic (CT) findings of Legionella pneumophila pneumonia. METHODS CT scans obtained from 23 sporadic cases of L. pneumophila pneumonia were retrospectively reviewed. Chest CT findings were analyzed with regard to the patterns and distributions of pulmonary abnormalities. We also analyzed the histopathology of lungs from guinea pigs with experimentally induced L. pneumophila pneumonia. RESULTS Consolidation and ground-glass opacity (GGO) were the main findings of CT scans in L. pneumophila pneumonia. The distribution of opacities was categorized as non-segmental (n=20) and segmental (n=4). Non-segmental distribution may follow an onset of segmental distribution. Pleural effusion was observed in 14 (58.3%) patients, of which 13 were accompanied with non-segmental distribution. Abscess formation was observed in only one immunocompromised patient. In the animal pneumonia model, the lesions comprised of terminal bronchioles, alveolar spaces, and interstitia. Small bacilli were observed to be contained by many macrophages within the alveoli. CONCLUSION Non-segmental distribution was significantly more frequent than segmental distribution in L. pneumophila pneumonia. It is possible that L. pneumophila infection initially results in segmental pneumonia, which progresses to typical non-segmental distribution.

Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

BackgroundLegionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells.MethodsNuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila.ResultsThe virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells.ConclusionInfection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.