Kaori Uezu

Critical role of Vα14+ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection

The present study was designed to elucidate the role of Vα14+ NKT cells in the host defense against pulmonary infection with Streptococcus pneumoniae using Jα281 gene‐disrupted mice (Jα281KO mice) that lacked this lymphocyte subset. In these mice, pneumococcal infection was severely exacerbated, as shown by the shorter survival time and marked increase of live bacteria in the lung compared to wild‐type (WT) mice. The proportion of Vα14+ NKT cells, detected by an α‐galactosylceramide (α‐GalCer)‐loaded CD1d tetramer, increased in thelung after S. pneumoniae infection. This increase was significantly reduced in mice with a genetic disruption of monocyte chemotactic protein (MCP)‐1, which was produced in the early phaseof infection in WT mice. In the lungs of Jα281KO mice, the number of neutrophils was significantly lower at 12 h than that in WT mice. In support of this finding, macrophage inflammatory protein (MIP)‐2 and TNF‐α synthesis in infected lungs was significantly reduced at 3 h and at both 3 and 6 h, respectively, in Jα281KO mice, compared to WT mice. In addition, treatment of mice with α‐GalCer significantly improved the outcome of this infection. Our results demonstrated MCP‐1‐dependent recruitment of Vα14+ NKT cells and their critical role in early host protection against S. pneumoniae by promoting the trafficking of neutrophils to the site of infection.

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.

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.

Lower expression of Th1‐related cytokines and inducible nitric oxide synthase in mice with streptozotocin‐induced diabetes mellitus infected with Mycobacterium tuberculosis

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin‐induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL‐12 and IFN‐γ in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL‐4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL‐12 and IFN‐γ. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL‐12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN‐γ upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD‐restimulated spleen cells reduced the synthesis of IFN‐γ only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1‐related cytokine synthesis. Our results suggest that the reduced production of Th1‐related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.

Accumulation of γδ T Cells in the Lungs and Their Regulatory Roles in Th1 Response and Host Defense against Pulmonary Infection with Cryptococcus neoformans

The present study was designed to elucidate the role of γδ T cells in the host defense against pulmonary infection with Cryptococcus neoformans. The γδ T cells in lungs commenced to increase on day 1, reached a peak level on day 3 or 6, and then decreased on day 10 after intratracheal infection. The increase of these cells was similar in monocyte chemoattractant protein (MCP)-1-deficient mice, although that of NK and NKT cells was significantly reduced. The number of live microorganisms in lungs on days 14 and 21 was significantly reduced in mice depleted of γδ T cells by a specific mAb compared with mice treated with control IgG. Similarly, elimination of this fungal pathogen was promoted in γδ T cell-deficient (TCR-δ−/−) mice compared with control littermate mice. Finally, lung and serum levels of IFN-γ on days 7 and 14 and on day 7 postinfection, respectively, were significantly higher in TCR-δ−/− mice than in littermate mice, whereas levels of TGF-β showed the opposite results. IL-4 and IL-10 were not different between these mice. IFN-γ production by draining lymph node cells upon restimulation with cryptococcal Ags was significantly higher in the infected TCR-δ−/− mice than in control mice. Our results demonstrated that γδ T cells accumulated in the lungs in a manner different from NK and NKT cells after cryptococcal infection and played a down-modulatory role in the development of Th1 response and host resistance against this fungal pathogen.

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.

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.

CpG oligodeoxynucleotides promote the host protective response against infection with Cryptococcus neoformans through induction of interferon-gamma production by CD4+ T cells.

In the present study, we elucidated the effect of synthetic CpG‐containing oligodeoxynucleotides (ODN) on pulmonary and disseminated infection caused by Cryptococcus neoformans. CDF‐1 mice were inoculated intratracheally with a highly virulent strain of this pathogen, which resulted in massive bacterial growth in the lung, dissemination to the brain and death. Administration of CpG‐ODN promoted the clearance of C. neoformans in the lungs, decreased their dissemination to brain and prolonged the survival of infected mice. These effects correlated well with the enhanced production of interleukin (IL)‐12 and interferon (IFN)‐γ and attenuated secretion of IL‐4 in bronchoalveolar lavage fluids (BALF) and promoted development of Th1 cells, as indicated by the increased production of IFN‐γ by paratracheal lymph node cells upon restimulation with cryptococcal antigens. The IFN‐γ synthesis in BALF was inhibited by depletion of CD8+ and CD4+ T cells on days 7 and 14 after infection, respectively, but not by depletion of NK and γδ T cells. Consistent with these data, intracellular expression of IFN‐γ was detected predominantly in CD8+ and CD4+ T cells in the lung on days 7 and 14, respectively. The protective effect of CpG‐ODN, as shown by the prolonged survival, was completely and partially inhibited by depletion of CD4+ or CD8+ T cells, respectively, but not by depletion of other cells. Finally, TNF‐α was markedly induced by CpG‐ODN, and the protective effect of this agent was strongly inhibited by neutralizing anti‐TNF‐α MoAb. Our results indicate that CpG‐ODN alters the Th1–Th2 cytokine balance and promotes host resistance against infection with C. neoformans.

Anti-CD11b Monoclonal Antibody Suppresses Brain Dissemination of Cryptococcus neoformans in Mice

To elucidate the role of the β2 integrin family of adhesion molecules in the disseminated infection of Cryptococcus neoformans from the lung to the central nervous system, we examined the effects of monoclonal antibodies (mAbs) against CD11a, CD11b, CD11c and CD18 on the number of live microorganisms in both the lung and brain of mice three weeks after intratracheal infection. Administration of anti‐CD11b mAb partially, but reproducibly, reduced the fungal loads in the brain in three independent experiments, while the lung loads were not affected. In addition, the same treatment significantly decreased the number of live microorganisms in the blood. In sharp contrast, the brain loads one week after intravenous injection with C. neoformans were not affected by treatment with anti‐CD11b mAb. Finally, administration of mAb against other adhesion molecules (CD11a, CD11c or CD18) failed to affect the fungal loads in the brain as well as in the lung three weeks after intratracheal instillation, except for anti‐CD18 mAb which rather increased the brain loads. Our results suggested that CD11b might be involved at least in part in the process of fungal dissemination from lung to brain, although the significance of other β2 integrin family adhesion molecules remains to be substantiated.