Masahiko Hatano

The role of autophagy during the early neonatal starvation period

At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3–12 h before returning to basal levels within 1–2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (∼ 12 h) is much shorter than that of wild-type mice (∼ 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of ‘self’ proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.

IL-23 and Th17 Cells Enhance Th2-Cell–mediated Eosinophilic Airway Inflammation in Mice

RATIONALE The IL-23-IL-17A-producing CD4(+) T-cell (Th17 cell) axis plays an important role in the development of chronic inflammatory diseases, including autoimmune diseases. However, the role of the IL-23-Th17 cell axis in the regulation of allergic airway inflammation is still largely unknown. OBJECTIVES To determine the role of IL-23 and Th17 cells in allergic airway inflammation. METHODS We examined the effect of anti-IL-23 antibody on antigen-induced airway inflammation. We also investigated the effect of enforced expression of IL-23 on allergic airway inflammation by generating lung-specific IL-23 transgenic mice. Moreover, we examined the effect of adoptive transfer of antigen-specific Th17 cells on allergic airway inflammation. MEASUREMENTS AND MAIN RESULTS IL-23 mRNA was expressed in the lung of sensitized mice upon antigen inhalation, and the neutralization of IL-23 decreased antigen-induced eosinophil recruitment and Th2 cytokine production in the airways. The enforced expression of IL-23 in the airways significantly enhanced antigen-induced eosinophil and neutrophil recruitment into the airways; Th2 cytokine, IL-17A, and tumor necrosis factor (TNF)-alpha production in the airways; goblet cell hyperplasia; and airway hyperresponsiveness. Moreover, IL-23-mediated enhancement of antigen-induced Th2 cytokine production and eosinophil recruitment in the airways was still observed in the mice lacking IL-17A. Furthermore, although adoptive transfer of antigen-specific Th17 cells alone induced neutrophil but not eosinophil recruitment into the airways upon antigen inhalation, cotransfer of Th17 cells with Th2 cells significantly enhanced antigen-induced Th2-cell-mediated eosinophil recruitment into the airways and airway hyperresponsiveness. CONCLUSIONS IL-23 and Th17 cells not only induce Th17-cell-mediated neutrophilic airway inflammation but also up-regulate Th2-cell-mediated eosinophilic airway inflammation.

Role for Bcl-6 in the generation and maintenance of memory CD8+ T cells

Naïve T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8+ T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8+ T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.

Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells.

After immunization with T cell-dependent antigens, the high-affinity B cells selected in germinal centers differentiate into memory B cells or long-lived antibody-forming cells. However, a role for germinal centers in development of these B lineage cells is still controversial. We show here that Bcl6-deficient B cells, which cannot develop germinal centers, differentiated into IgM and IgG1 memory B cells in the spleen but barely differentiated into long-lived IgG1 antibody-forming cells in the bone marrow. Mutation in the V-heavy gene was null in these memory B cells. Therefore, Bcl6 and germinal center formation are essential for somatic hypermutation, and generation of memory B cells can occur independently of germinal center formation, somatic hypermutation, and Ig class switching.

Transcriptional activation of survivin through the NF‐κB pathway by human T‐cell leukemia virus type I tax

Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. We previously reported the survivin expression profile in ATL, a CD4‐positive T‐cell malignancy caused by HTLV‐I. HTLV‐I Tax is thought to play an important role in immortalization of T cells. We have shown also that the expression of Tax protected the mouse T‐cell line CTLL‐2 against apoptosis induced by deprivation of IL‐2 and converted its growth from being IL‐2 dependent to being IL‐2 independent through the NF‐κB pathway. In our study, we demonstrate that constitutive expression of survivin was associated with resistance to apoptosis after IL‐2 deprivation in Tax‐expressing CTLL‐2 cells. Transient transfection assays showed that survivin promoter was transactivated by Tax, via the activation of NF‐κB. Pharmacological NF‐κB inhibition resulted in suppression of survivin expression and caused apoptosis of Tax‐expressing CTLL‐2 cells. Our findings suggest that activated NF‐κB signaling contributes directly to malignant progression of ATL by preventing apoptosis, acting through the prosurvival protein survivin.

Prostaglandin D2 Reinforces Th2 Type Inflammatory Responses of Airways to Low-dose Antigen through Bronchial Expression of Macrophage-derived Chemokine

PGD2, a lipid mediator released from mast cells, is known to participate in allergic reactions. However, the mechanism by which PGD2 contributes to such reactions remains unclear. We established a novel experimental model of asthma that permitted direct assessment of the role of PGD2 in airway inflammation. Antigen-sensitized mice were exposed to aerosolized prostaglandin D2 (PGD2) 1 d before challenge with low-dose aerosolized antigen. Not only the numbers of eosinophils, lymphocytes, and macrophages but also the levels of IL-4 and IL-5 in bronchoalveolar lavage fluid were higher in PGD2-pretreated mice than in control mice. The expression of macrophage-derived chemokine (MDC), a chemoattractant for Th2 cells, was greater in PGD2-pretreated mice than in control. Injection of anti-MDC antibody into PGD2-pretreated mice markedly inhibited inflammatory cell infiltration as well as Th2 cyto-kine production after antigen challenge. These results indicate that PGD2 accelerates Th2 type inflammation by induction of MDC. Our results suggest that this mechanism may play a key role in the development of human asthma and that MDC might be a target molecule for therapeutic intervention.

A novel pathogenesis of megacolon in Ncx/Hox11L.1 deficient mice.

The Ncx/Hox11L.1 gene, a member of the Hox11 homeobox gene family, is mainly expressed in neural crest-derived tissues. To elucidate the role of Ncx/Hox11L.1, the gene has been inactivated in embryonic stem cells by homologous recombination. The homozygous mutant mice were viable. These mice developed megacolon with enteric ganglia by age 3-5 wk. Histochemical analysis of the ganglia revealed that the enteric neurons hyperinnervated in the narrow segment of megacolon. Some of these neuronal cells degenerated and neuronal cell death occurred in later stages. We propose that Ncx/Hox11L.1 is required for maintenance of proper functions of the enteric nervous system. These mutant mice can be used to elucidate a novel pathogenesis for human neuronal intestinal dysplasia.

Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation

Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD(-/-)) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1(-/-) ES cells and Pkd1(+/+) morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1(-/-) and Pkd1(+/+) renal tubular epithelial cells in the early stages of cystogenesis. Pkd1(-/-) cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1(+/+) cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1(-/-) cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1(-/-) mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.

DNA polymerase θ is preferentially expressed in lymphoid tissues and upregulated in human cancers

DNA polymerase θ (Pol θ) is a recently identified family A polymerase that contains an intrinsic helicase domain. Drosophila Pol θ mutants are hypersensitive to bifunctional DNA crosslinking agents and exhibit an elevated frequency of spontaneous chromosomal aberrations, suggesting a role for Pol θ in repair of DNA interstrand crosslinks and in the general maintenance of genome stability. To investigate a possible involvement of Pol θ in tumorigenesis, we have examined its expression in various normal and malignant tissues. Paired tumor and adjacent nontumorous tissues from patients with lung (n = 27), stomach (n = 28) and colon (n = 26) cancer, as well as 26 normal human tissues, were examined for Pol θ expression by RT‐PCR, Northern or Western blot analysis. Pol θ was predominantly expressed in primary lymphoid organs including the fetal liver, thymus and bone marrow where lymphocyte progenitors undergo V(D)J rearrangements of their antigen receptor genes. In addition, Pol θ expression was upregulated in germinal center B cells, in which class switch recombination of the immunoglobulin genes occurs. Examination of Pol θ expression in matched cancer specimens revealed that Pol θ was barely detectable in the nontumorous tissues but was upregulated in 17 of 27 (63%) lung, 11 of 28 (39%) stomach and 20 of 26 (77%) colon cancers. Moreover, patients with high levels of Pol θ expression had a significantly poorer clinical outcome compared with those expressing low levels of Pol θ. These results implicate that Pol θ may have a specialized function in lymphocytes and that its overexpression may contribute to tumor progression.

A Role for c-fos/Activator Protein 1 in B Lymphocyte Terminal Differentiation

Expression of B lymphocyte-induced maturation protein 1 (Blimp-1) transcription factor is essential for promoting B cell differentiation into plasma cells. However, a critical transcription factor for Blimp-1 expression in activated B cells is unclear. When splenic B cells were stimulated with CD40 ligand (CD40L) and IL-4, terminal differentiation was induced in the B cells from c-fos transgenic (H2-c-fos) mice but barely in those from control littermates and from c-fos-deficient mice. AP-1 family and Blimp-1 mRNAs were transiently induced in the control B cells, and overexpression of c-Fos induced a sufficient amount of Blimp-1 for terminal differentiation in the H2-c-fos B cells. When normal and c-fos-deficient B cells were stimulated with LPS, a sufficient amount of Blimp-1 for terminal differentiation was induced in those B cells. However, expression of c-fos/AP-1 family mRNAs in LPS-stimulated normal B cells was similar to that of normal B cells stimulated with CD40L and IL-4. EMSA and chromatin immunoprecipitation assays using the AP-1-binding DNA sequence in the murine Blimp-1 promoter region demonstrated that AP-1-binding activity in nuclear protein of LPS-stimulated normal B cells was prolonged more than that in normal B cells stimulated with CD40L and IL-4. Furthermore, the percentage of CD138+ B cells within germinal center B cells in the spleen and the number of Ab-forming cells in the bone marrow of H2-c-fos mice was larger than that of control mice 12 days after immunization. Thus, although c-Fos is not essential for Blimp-1 expression, c-Fos/AP-1 positively regulates Blimp-1 expression and terminal differentiation of activated B cells.