Yuki Kinjo

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.u2004pneumoniae 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 12u2004h 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 3u2004h and at both 3 and 6u2004h, 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.u2004pneumoniae 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.

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.

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.

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.

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+u2003and 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+u2003and 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+u2003or 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.

Circulating Soluble CD4 Directly Prevents Host Resistance and Delayed-Type Hypersensitivity Response to Cryptococcus neoformans in Mice

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed‐type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane‐bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti‐CD4 mAb restored the attenuated host defense. Second, in CD4 gene‐disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene‐transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock‐transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti‐CD4 mAb. Finally, serum levels of IFN‐γ, IL‐12 and IL‐18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL‐4 and IL‐10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1‐type cytokines.