Mana Miyakoda

Radiation-Induced Senescence-like Growth Arrest Requires TP53 Function but not Telomere Shortening

Abstract Suzuki, K., Mori, I., Nakayama, Y., Miyakoda, M., Kodama, S. and Watanabe, M. Radiation-Induced Senescence-like Growth Arrest Requires TP53 Function but not Telomere Shortening. Normal human diploid cells irradiated with X rays showed permanent cell cycle arrest and exhibited senescence-like phenotypes including the expression of senescence-associated β-galactosidase (SA-β-gal). X irradiation caused persistent phosphorylation of TP53 at Ser 15 and accumulation of the TP53 protein, followed by the induction of CDKN1A (also known as p21Waf1/Cip1) and CDKN2A (also known as p16), preceded the expression of SA-β-gal. NCI-H1299 human lung carcinoma cells, in which no TP53 protein was expressed, were irradiated with X rays with or without the exogenous expression of TP53 gene. Although induction of TP53 protein alone could induce SA-β-gal expression, the frequency of SA-β-gal-positive cells was significantly increased when TP53-induced H1299 cells were exposed to X rays. The mean terminal restriction fragment length in normal human cells was approximately 12 kb and did not change in SA-β-gal-positive cells. These results indicate that ionizing radiation induces senescence-like growth arrest that is dependent on TP53 function but independent of telomere shortening. Our findings suggest that cells harboring irreparable DNA damage are programmed to undergo premature senescence to maintain the integrity of the genome.

Malaria-Specific and Nonspecific Activation of CD8+ T Cells during Blood Stage of Plasmodium berghei Infection

Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8+ T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8+ T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8+ T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8+ T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8+ T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8+ T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8+ T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.

Activation of ATM and phosphorylation of p53 by heat shock.

p53 protein is phosphorylated in response to various stresses. Here we examined phosphorylation of p53 protein in normal human diploid cells after heat shock at 43°C for 2 h. We found that heat shock stimulates phosphorylation of p53 at Ser15 but not at Ser20, while X-irradiation at 4 Gy and 10 J/m2 of UV induces phosphorylation of p53 at Ser15 and less significantly at Ser20. Increased phosphorylation of Ser15 was also observed in heat shocked GM638, the SV40-transformed human fibroblast cell line. Although X-ray irradiation induced phosphorylation of Ser6, 9, 20, and 37 in GM638 cells, heat shock did not affect the phosphorylation level of these serines. We observed little or no phosphorylation of p53 at Ser15 in two primary ataxia telangiectasia fibroblast cells, that are defective in ATM. Using an in vitro kinase assay, we confirmed that immunoprecipitated ATM from both heat-shocked and X-irradiated normal human diploid cells can phosphorylate p53 at Ser15 to a similar extent. These results indicate that heat shock induces phosphorylation of p53, especially at Ser15, and its phosphorylation is mediated by ATM kinase.

Interferon regulatory factor 4 differentially regulates the production of Th2 cytokines in naïve vs. effector/memory CD4+ T cells

Interferon regulatory factor (IRF) 4 is a member of the IRF family of transcription factors and plays critical roles in the development of CD4+ T cells into Th2 and Th17 cells. Using the infection model of Nippostrongyrus brasiliensis, we have confirmed the critical roles of IRF-4 in Th2 development in vivo by using IRF-4−/− BALB/c mice. However, naïve IRF-4−/−CD4+ T cells produced Th2 cytokines, including IL-4, IL-5, and IL-10, but not IL-2 or IFN-γ, at levels higher than wild-type BALB/c CD4+ T cells in response to T cell receptor stimulation. In contrast, effector/memory IRF-4−/−CD4+ T cells did not exhibit increased production of Th2 cytokines. Knockdown of IRF-4 expression by using small interfering RNA promoted IL-4 production in naïve CD4+ T cells but inhibited it in effector/memory CD4+ T cells. These results indicate that IRF-4 plays differential roles in the regulation of Th2 cytokine production in naïve CD4+ T cells and effector/memory CD4+ T cells. IRF-4 inhibits Th2 cytokine production in naïve CD4+ T cells, whereas it promotes Th2 cytokine production in effector/memory CD4+ T cells.

Heat shock induces centrosomal dysfunction, and causes non-apoptotic mitotic catastrophe in human tumour cells

Normal human diploid cells and various human tumour cells were heat shocked at 43°C for 2h and allowed to recover at 37°C. It was found that heat shock treatment transiently disrupted the immunostaining of centrosomes, and no centrosome staining was detected in either normal or tumour cells 24h after heat shock. Staining recovered thereafter in normal cells, but in tumour cells abnormal centrosomes, multiple and minute centrosomes were induced. While normal cells were arrested in G1 and G2 after heat shock, significant numbers of mitotic cells with multiple poles appeared in tumour cells. Subsequently, cells with multiple micronuclei increased in tumour cells with time after heat shock. Although the nuclear morphology of these cells was similar to that of the apoptotic cells, no DNA ladder formation was observed up to 4 days after heat shock. Furthermore, an in situ assay failed to detect signals representative of apoptosis, indicating that apoptosis did not appear to be involved in heat shock-induced cell death of human tumour cells. Instead, cell lethality was associated with mitotic catastrophe.

CD8+ T Cells Specific for a Malaria Cytoplasmic Antigen Form Clusters around Infected Hepatocytes and Are Protective at the Liver Stage of Infection

ABSTRACT Following Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8+ T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8+ T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8+ T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8+ T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8+ T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8+ T cells during liver-stage infection.

IRF4 in Dendritic Cells Inhibits IL-12 Production and Controls Th1 Immune Responses against Leishmania major

IRF4 is a transcription factor from the IRF factor family that plays pivotal roles in the differentiation and function of T and B lymphocytes. Although IRF4 is also expressed in dendritic cells (DCs) and macrophages, its roles in these cells in vivo are not clearly understood. In this study, conditional knockout mice that lack IRF4 in DCs or macrophages were generated and infected with Leishmania major. Mice lacking DC expression of IRF4 showed reduced footpad swelling compared with C57BL/6 mice, whereas those lacking IRF4 in macrophages did not. Mice with IRF4-deficient DCs also showed reduced parasite burden, and their CD4+ T cells produced higher levels of IFN-γ in response to L. major Ag. In the draining lymph nodes, the proportion of activated CD4+ T cells in these mice was similar to that in the control, but the proportion of IFN-γ–producing cells was increased, suggesting a Th1 bias in the immune response. Moreover, the numbers of migrating Langerhans cells and other migratory DCs in the draining lymph nodes were reduced both before and postinfection in mice with IRF4 defects in DCs, but higher levels of IL-12 were observed in IRF4-deficient DCs. These results imply that IRF4 expression in DCs inhibits their ability to produce IL-12 while promoting their migratory behavior, thus regulating CD4+ T cell responses against local infection with L. major.

Daily Feeding of Fructooligosaccharide or Glucomannan Delays Onset of Senescence in SAMP8 Mice

We hypothesized that daily intake of nondigestible saccharides delays senescence onset through the improvement of intestinal microflora. Here, we raised senescence accelerated mice prone 8 (SAMP8) on the AIN93 diet (CONT), with sucrose being substituted for 5% of fructooligosaccharide (FOS) or 5% of glucomannan (GM), 15 mice per group. Ten SAMR1 were raised as reference of normal aging with control diet. Grading of senescence was conducted using the method developed by Hosokawa, and body weight, dietary intake, and drinking water intake were measured on alternate days. Following 38 weeks of these diets we evaluated learning and memory abilities using a passive avoidance apparatus and investigated effects on the intestinal microflora, measured oxidative stress markers, and inflammatory cytokines. Continuous intake of FOS and GM significantly enhanced learning and memory ability and decelerated senescence development when compared with the CONT group. Bifidobacterium levels were significantly increased in FOS and GM-fed mice. Urinary 8OHdG, 15-isoprostane, serum TNF-α, and IL-6 were also lower in FOS-fed mice, while IL-10 in FOS and GM groups was higher than in CONT group. These findings suggest that daily intake of nondigestible saccharides delays the onset of senescence via improvement of intestinal microflora.

Interleukin-27-Producing CD4(+) T Cells Regulate Protective Immunity during Malaria Parasite Infection.

Interleukin-27 (IL-27) is a heterodimeric regulatory cytokine of the IL-12 family, which is produced by macrophages, dendritic cells, and B cells upon stimulation through innate immune receptors. Here, we described regulatory CD4(+) T cells that produce IL-27 in response to T cell receptor stimulation during malaria infection, inhibiting IL-2 production and clonal expansion of other T cells in an IL-27-dependent manner. IL-27-producing CD4(+) T cells were Foxp3(-)CD11a(+)CD49d(+) malaria antigen-specific CD4(+) T cells and were distinct from interferon-γ (IFN-γ) producing Th1 or IL-10 producing Tr1 cells. In mice lacking IL-27 in T cells, IL-2 production was restored and clonal expansion and IFN-γ production by specific CD4(+) T cells were improved, culminating in reduced parasite burden. This study highlights a unique population of IL-27 producing regulatory CD4(+) T cells and their critical role in the regulation of the protective immune response against malaria parasites.

The species specificity of immunity generated by live whole organism immunisation with erythrocytic and pre-erythrocytic stages of rodent malaria parasites and implications for vaccine development.

A promising strategy for the development of a malaria vaccine involves the use of attenuated whole parasites, as these present a greater repertoire of antigens to the immune system than subunit vaccines. The complexity of the malaria parasites life cycle offers multiple stages on which to base an attenuated whole organism vaccine. An important consideration in the design and employment of such vaccines is the diversity of the parasites that are infective to humans. The most valuable vaccine would be one that was effective against multiple species/strains of malaria parasite. Here we compare the species specificity of pre-erythrocytic and erythrocytic whole organism vaccination using live parasites with anti-malarial drug attenuation. The cross-stage protection afforded by each vaccination strategy, and the possibility that immunity against one stage may be abrogated by exposure to other stages of both homologous and heterologous parasites was also assessed. The rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium vinckei lentum are to address these questions, as they offer the widest possible genetic distance between sub-species of malaria parasites infectious to rodents. It was found that both erythrocytic and pre-erythrocytic stage immunity generated by live, attenuated parasite vaccination have species-specific components, with pre-erythrocytic stage immunity offering a much broader pan-species protection. We show that the protection achieved following sporozoite inoculation with concurrent mefloquine treatment is almost entirely dependent of CD8(+) T-cells. Evidence is presented for cross-stage protection between erythrocytic and pre-erythrocytic stage vaccination. Finally, it is shown that, with these species, an erythrocytic stage infection of either a homologous or heterologous species following immunisation with pre-erythrocytic stages does not abrogate this immunity. This is the first direct comparison of the specificity and efficacy of erythrocytic and pre-erythrocytic stage whole organism vaccination strategies utilising the same parasite species pair.