Roles of pigment epithelium-derived factor in exercise-induced suppression of senescence and its impact on lung pathology in mice.

Senescent cells contribute to tissue aging and underlie the pathology of chronic diseases. The benefits of eliminating senescent cells have been demonstrated in several disease models, and the efficacy of senolytic drugs is currently being tested in humans. Exercise training has been shown to reduce cellular senescence in several tissues; however, the mechanisms responsible remain unclear. We found that myocyte-derived factors significantly extended the replicative lifespan of fibroblasts, suggesting that myokines mediate the anti-senescence effects of exercise. A number of proteins within myocyte-derived factors were identified by mass spectrometry. Among these, pigment epithelium-derived factor (PEDF) exerted inhibitory effects on cellular senescence. Eight weeks of voluntary running increased Pedf levels in skeletal muscles and suppressed senescence markers in the lungs. The administration of PEDF reduced senescence markers in multiple tissues and attenuated the decline in respiratory function in the pulmonary emphysema mouse model. We also showed that blood levels of PEDF inversely correlated with the severity of COPD in patients. Collectively, these results strongly suggest that PEDF contributes to the beneficial effects of exercise, potentially suppressing cellular senescence and its associated pathologies.

Chimeric flavivirus causes vascular leakage and bone marrow suppression in a mouse model.

Previously, we demonstrated the utility of a recombinant chimeric flavivirus (DV2ChimV), which carries the premembrane (prM) and envelope (E) genes of a type 2 DENV clinical (Thai) isolate on a backbone of Japanese encephalitis virus, for evaluating the protective efficacy of antidengue envelope antibodies both in vitro and in vivo. Here, to assess the potential use of this model for pathological studies, we aimed to characterize interferon-α/ß-γ-receptor double-knockout mice (IFN-α/ß/γR dKO mice) infected with DV2ChimV. Vascular leakage and bone marrow suppression are unique features of severe dengue. In the current model, DV2ChimV caused vascular leakage in the liver and intestine at the moribund stage. High levels of virus were detected in the bone marrow, and strong bone marrow suppression (i.e., disappearance of megakaryocytes and erythroblastic islets) was observed. These observations suggest that the DV2ChimV-infected mouse model mimics the vascular leakage and bone marrow suppression observed in human cases.

Age-related changes in lung function in National Center for Geriatrics and Gerontology Aging Farm C57BL/6N mice.

Aging is an extremely complex biological process, and various models, from unicellular organisms to mammals, have been used in its research. The mouse is the most widely used model for studying human aging and diseases due to its high homology and well-established strategies for genetic manipulation. Despite these advantages, the maximum lifespan of laboratory mice is nearly three years, which makes it time-consuming to obtain animals of the desired age. To avoid this issue and efficiently conduct aging research, the National Center for Geriatrics and Gerontology operates its "Aging Farm", a system that supplies aged animals in response to researchers' requests. In the present study, as part of the Aging Farm project, we examined changes in the physiological functions of the lungs and gene expression in lung tissues of Aging Farm animals as they aged. A decline in the physiological function of the lungs was already apparent before 6 months of age, and it continued until at least 1 year of age. On the other hand, gene expression profiling by RNA sequencing showed small changes in the early stages of aging but more pronounced changes at 12 and 24 months of age than at 3 months of age. Age-related lung tissue changes are considered to be involved in the pathogenesis of various chronic respiratory diseases, and the characterization of animals as they age will ensure the quality of the Aging Farm as a resource for aging research.

Protocol for assessing senescence-associated lung pathologies in mice.

Cellular senescence underlies tissue aging and aging-associated pathologies, as well as lung pathology. We and others have shown that elimination of senescent cells alleviates pulmonary diseases such as fibrosis and emphysema in animal models. We herein describe a protocol for assessing senescence-dependent lung phenotypes in mice. This protocol describes the use of ARF-DTR mice for semi-genetic elimination of lung senescent cells, followed by a pulmonary function test and the combination with pulmonary disease models to study lung pathologies. For complete details on the use and execution of this protocol, please refer to Hashimoto et al. (2016), Kawaguchi et al. (2021), and Mikawa et al. (2018).

Cellular senescence promotes cancer metastasis by enhancing soluble E-cadherin production.

Cellular senescence acts as a potent tumor-suppression mechanism in mammals; however, it also promotes tumor progression in a non-cell-autonomous manner. We provided insights into the mechanism underlying senescence-dependent metastatic cancer development. The elimination of senescent cells suppressed the lung metastasis of melanoma cells. Using an antibody array screening of humoral factor(s) that depend on cellular senescence, we identified soluble E-cadherin (seCad) as a potential mediator of the senescence-induced melanoma metastasis. seCad enhanced the invasive activity of melanoma cells both in vitro and in vivo, and gene expression profiling revealed that seCad induced genes associated with poor prognosis in patients with melanoma. An analysis of sera from patients revealed that serum seCad is associated with distant metastasis. Our data suggest that senescent cells promote metastatic lung cancer through seCad, and that seCad may be a potential diagnostic marker as well as a therapeutic target for metastatic lung cancer.

Chimeric flavivirus enables evaluation of antibodies against dengue virus envelope protein in vitro and in vivo.

In a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/ß-γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.

Elimination of p19ARF-expressing cells enhances pulmonary function in mice.

Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells.

Loss of HuR leads to senescence-like cytokine induction in rodent fibroblasts by activating NF-κB.

BACKGROUND: HuR (human antigen R) is a ubiquitously expressed member of the Hu/ELAV family of proteins that is involved in diverse biological processes. HuR has also been shown to play an important role in cell cycle arrest during replicative senescence in both human and mouse cells. Senescent cells not only halt their proliferation, but also activate the secretion of proinflammatory cytokines. A persistent DNA damage response is essential for the senescence-associated secretory phenotype (SASP), and increasing evidence has suggested that the SASP is associated with malignancy. METHODS: Senescence-associated phenotypes were analyzed in MEFs and other cell line in which HuR expression is inhibited by sh-RNA-mediated knockdown. RESULTS: RNAi-mediated HuR inhibition resulted in an increase in SASP-related cytokines. The induction of SASP factors did not depend on ARF-p53 pathway-mediated cell cycle arrest, but required NF-κB activity. In the absence of HuR, cells were defective in the DNA-damage response, and single strand DNA breaks accumulated, which may have caused the activation of NF-κB and subsequent cytokine induction. CONCLUSIONS: In the absence of HuR, cells exhibit multiple senescence-associated phenotypes. Our findings suggest that HuR regulates not only the replicative lifespan, but also the expression of SASP-related cytokines in mouse fibroblasts. GENERAL SIGNIFICANCE: RNA-binding protein HuR protects cells from undergoing senescence. Senescence-associated phenotypes are accelerated in HuR-deficient cells.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.

Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection.

The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals.

Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells.

BACKGROUND: We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cells after immunization with T-cell-dependent antigen. Zizimin2 was revealed to be a new family member of Dock (dedicator of cytokinesis), Dock11, which is the guanine nucleotide exchange factor for Cdc42, a low-molecular-weight GTPase. However, the molecular function of Zizimin2 in acquired immunity has not been elucidated. RESULTS: In this study, we show that the protein expression of Zizimin2, which is also restricted to lymphoid tissues and lymphocytes, is reduced in aged mice. Over-expression of full-length Zizimin2 induced filopodial formation in 293T cells, whereas expression of CZH2 domain inhibited it. Stimulation of Fcγ receptor and Toll-like receptor 4 triggered Zizimin2 up-regulation and Cdc42 activation in bone marrow-derived dendritic cells. CONCLUSIONS: These data suggest that Zizimin2 is an immune-related and age-regulated guanine nucleotide exchange factor, which facilitates filopodial formation through activation of Cdc42, which results in activation of cell migration.

Effectiveness of BCG vaccination to aged mice.

BACKGROUND: The tuberculosis (TB) still increases in the number of new cases, which is estimated to approach 10 million in 2010. The number of aged people has been growing all over the world. Ageing is one of risk factors in tuberculosis because of decreased immune responses in aged people. Mycobacterium bovis Bacillus Calmette Guérin (BCG) is a sole vaccine currently used for TB, however, the efficacy of BCG in adults is still a matter of debate. Emerging the multidrug resistant Mycobacterium tuberculosis (MDR-TB) make us to see the importance of vaccination against TB in new light. In this study, we evaluated the efficacy of BCG vaccination in aged mice. RESULTS: The Th1 responses, interferon-γ production and interleukin 2, in BCG inoculated aged mice (24-month-old) were comparable to those of young mice (4- to 6-week-old). The protection activity of BCG in aged mice against Mycobacterium tuberculosis H37Rv was also the same as young mice. CONCLUSION: These findings suggest that vaccination in aged generation is still effective for protection against tuberculosis.

Rat monoclonal antibodies against new guanine nucleotide exchange factor, mouse Zizimin2.

We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cell after immunization with T-cell-dependent antigen. Zizimin2 was revealed that it is a new family member of Dock (dedicator of cytokinesis), Dock11, which was the guanine nucleotide exchange factor for Cdc42, the Rho family of low molecular weight GTPases. However, neither the molecular function of Zizimin2 in the acquired immunity nor the physiological role in decrease of immunological activity along with aging has been known. To facilitate the further analysis of physiological function of this molecule, we have established rat monoclonal antibodies (MAbs) against mouse Zizimin2 protein. Recombinant His-tagged partial mouse Zizimin2 protein was expressed in Escherichia coli, purified, and applied as an antigen for immunization. The desired hybridomas were selected by the recombinant Zizimin2 protein coated enzyme-linked immunosorbent assay screening, and we generated stable hybridoma cell lines that produced the antibody against murine Zizimin2 protein. We determined their isotypes and further examined capability or availability in immunoblotting, immunoprecipitation, or immunofluorescence microscopy. Here we demonstrated the several appropriate antibodies for immunoblotting and immunofluorescence microscopy. These MAbs should therefore be very useful tools for study of the characterization of the Zizimin2 protein and the understanding of the biological function of Zizimin2-mediated immunosenescence.

Production of rat monoclonal antibodies against RNA-binding protein Hzf.

The hematopoietic zinc finger protein, Hzf, is induced in response to DNA damage or by Arf tumor suppressor in a p53-dependent manner. Recent studies have revealed that Hzf is an RNA-binding protein that regulates localization and translation of specific mRNA. The RNA-binding activity of Hzf is required for the functions of cerebellar purkinje cells and adipocytes, although their molecular mechanisms underlying the mRNA regulation largely remain unknown. To further investigate the molecular function of Hzf, we raised two rat monoclonal antibodies (MAb) against a peptide corresponding to the C-terminal region of the mouse/human Hzf protein. Both MAbs reacted with the native protein expressed in mammalian cells, and were highly efficient in detecting endogenous Hzf by immunoblotting, immunoprecipitation, and immunofluorescence. These MAbs should therefore be useful for further analysis of molecular functions of the Hzf protein and for identification of Hzf-binding proteins.

Large defects of type I allergic response in telomerase reverse transcriptase knockout mice.

Telomerase is critically important for the maintenance of a constant telomere length, which in turn, is related to the concepts of longevity and oncogenesis. In addition, it has been well documented that telomerase activity is expressed in immune cells in a highly regulated manner. We have studied systemic anaphylaxis in mouse telomerase reverse transcriptase knockout (mTERT(-/-)) mice to understand the significance of telomerase activity and telomere stability in mast cells, which induce a type I allergic response. Compared with wild-type mice, mTERT(-/-) mice displayed largely attenuated, IgE-mediated, passive anaphylactic responses, which were observed even in the early generations of mTERT(-/-) mice, and had decreased numbers of mast cells in vivo and impaired development of bone marrow-derived mast cells (BMMCs) induced by IL-3 or stem cell factor in vitro. Moreover, in mTERT(-/-) mice, BMMCs exhibited a large morphology and low proliferation rate, while they possessed a comparable degranulation capacity and cell surface expression level of c-kit and FcepsilonRI. These findings imply that telomerase activity has a definitive impact on the type I allergic response by altering the character of effecter mast cells.

Death-associated protein 3 regulates cellular senescence through oxidative stress response.

Death-associated protein 3 (DAP3) has been originally identified as a positive mediator of apoptosis. It has been revealed recently that the predominant localization of DAP3 to mitochondria implies its functional involvement in mitochondrial metabolism in addition to apoptosis. However, little is known about the molecular basis of these physiological functions of DAP3. Here, we demonstrate that DAP3 is reduced in both replicative and premature senescence induced by oxidative stress, and the DAP3 reduction induced by oxidative stress is observed mostly in a mitochondrial fraction. Using DAP3-specific short hairpin RNA (shRNA) in a clonogenic survival assay, we reveal that reduction of DAP3 induces resistance to oxidative stress and decreases intracellular reactive oxygen species (ROS) production. Furthermore, this strategy allows us to show that loss of DAP3 is involved in the avoidance of replicative senescence in mouse embryonic fibroblasts (MEFs). Thus, our study offers an insight into the potential regulatory function of mitochondrial DAP3 involved in cellular senescence.

Transcriptional regulation of ILT family receptors.

Ig-like transcripts (ILT/leukocyte Ig-like receptor/monocyte/macrophage Ig-like receptor or CD85) are encoded on human chromosome 19q13.4, designated the human leukocyte receptor complex, and are predominantly expressed on myeloid lineage cells. We investigated the transcriptional regulation of ILT1, ILT2, and ILT4 genes to elucidate control mechanisms operating on the specific expression of ILT receptors. Inhibitory ILT2 and ILT4 both have a similar genomic structure, in which the approximately 160-bp 5'-flanking regions function as core promoters with critically important PU.1 binding sites. However, an Sp1 family-binding GC-box is more influential in trans-activation of ILT2 than ILT4. Additionally, ILT4 transcription is tightly regulated by chromatin modifications accompanied by histone acetylation, which strictly controls expression within myeloid lineage cells. Activating ILT1 carries a core promoter corresponding to the intronic region of ILT2 and ILT4, where PU.1 and Runx1 binding sites are essential, but a downstream heat shock element also augments promoter activity. Thus, each ILT is regulated by a distinct transcriptional mechanism, although PU.1 acts as a common trans-acting factor. We also found that human CMV infection strongly trans-activates inhibitory ILT2 and ILT4 genes through the expression of immediate-early proteins.

Dramatic increase of telomerase activity during dendritic cell differentiation and maturation.

Telomerase, the reverse transcriptase that maintains telomere DNA, is usually undetectable in most adult tissues but is positive in embryonic tissues and in cancers. In addition, freshly islolated or in vitro-activated lymphocytes were shown to express high levels of telomerase activity, although its expression in myeloid cells including dendritic cells (DCs) is largely unknown. Here, we investigated telomerase activity during the differentiation and maturation process of DCs. In vitro culture of bone marrow (BM) cells with granulocyte macrophage-colony stimulating factor and interleukin-4 induced a dramatic increase of telomerase activity accompanied with their differentiation into DCs. Furthermore, stimulation with microbial components such as lipopolysaccharide (LPS), which triggers maturation of DCs, augmented the activity. In vivo responses of telomerase activity were also observed in splenic DCs by injection of LPS intraperitoneally. It is interesting that in old mice, telomerase activity of splenic DCs was significantly higher than young mice but rather decreased after LPS stimulation. By measuring expression of cell-surface activation markers, splenic DCs of old mice responded poorly to LPS stimulation. Such poor responses to LPS were also observed in BM-derived DCs. These different features of DCs between young and old mice may contribute to a pathogenesis to microbial infections.

Osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in DAP12-deficient mice.

Deletions in the DAP12 gene in humans result in Nasu-Hakola disease, characterized by a combination of bone fractures and psychotic symptoms similar to schizophrenia, rapidly progressing to presenile dementia. However, it is not known why these disorders develop upon deficiency in DAP12, an immunoreceptor signal activator protein initially identified in the immune system. Here we show that DAP12-deficient (DAP12(-/-)) mice develop an increased bone mass (osteopetrosis) and a reduction of myelin (hypomyelinosis) accentuated in the thalamus. In vitro osteoclast induction from DAP12(-/-) bone marrow cells yielded immature cells with attenuated bone resorption activity. Moreover, immature oligodendrocytes were arrested in the vicinity of the thalamus, suggesting that the primary defects in DAP12(-/-) mice are the developmental arrest of osteoclasts and oligodendrocytes. In addition, the mutant mice also showed synaptic degeneration, impaired prepulse inhibition, which is commonly observed in several neuropsychiatric diseases in humans including schizophrenia, and aberrant electrophysiological profiles in the thalami. These results provide a molecular basis for a unique combination of skeletal and psychotic characteristics of Nasu-Hakola disease as well as for schizophrenia and presenile dementia.