Effect of age on orthodontic tooth movement in mice.

Background/purpose: The number of middle-aged and elderly orthodontic patients is increasing due to changes in age composition. It is important to investigate the detailed mechanisms of bone remodeling in orthodontic tooth movement (OTM) in the elderly. However, there are few reports on the mechanism of tooth movement in the elderly. The purpose of the present study was to analyze OTM and osteoclastogenesis in aged mice and to elucidate the mechanism. Materials and methods: It has been reported that tumor necrosis factor (TNF)-α plays an important role in osteoclast formation and OTM. First, 8-week-old and 78-week-old male C57BL/6J mice were subcutaneously injected with TNF-α into the calvaiae, and micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and real-time PCR were performed to evaluate osteoclast formation and bone resorption. Furthermore, osteoclastogenesis by TNF-α and receptor activator of nuclear factor-kappa B ligand (RANKL) using bone marrow cells was evaluated in vitro. Finally, a nickel-titanium closed-coil spring was attached, mesial movement of the maxillary left first molar was performed, and tooth movement distance and osteoclast formation were evaluated. Results: Compared to 8-week-old mice, 78-week-old mice had decreased TNF-α-induced bone resorption, osteoclastogenesis, and TRAP and cathepsin K expression in the calvariae. In vitro osteoclast formation also decreased in 78-week-old mice. Furthermore, tooth movement distance and osteoclastogenesis were reduced. Conclusion: OTM decreased in aged mice, which was shown to be caused by a decrease in osteoclastogenesis. Therefore, it was suggested that it is necessary to keep in mind that tooth movement may be suppressed when treating elderly patients.

Toward the development of a vibrant, super-aged society: The future of medicine and society in Japan.

BACKGROUND: As Japan's population continues to age, it is estimated that the number of people aged ≥75 years will exceed 20 million by 2025. Furthermore, over the past 10 years, we have not reduced the difference between life expectancy and healthy life expectancy. Therefore, the extension of healthy life expectancy and the development of a healthy society are the most urgent issues. In terms of medical care, the changing times have inevitably led to changes in disease structures and medical demands; therefore, the medical delivery system has had to be changed to meet these demands. As dementia rapidly increases, it is important to address "frailty," a condition in which people become more vulnerable to environmental factors as they age, and there is a need to provide services to older people, particularly the old-old, that emphasize quality of life in addition to medical care. To realize a super-aged society that will remain vigorous and vibrant for many years, we need to rethink the future of Japanese medicine and healthcare, and the state of society. CURRENT SITUATION AND PROBLEMS: Disparity between healthy life expectancy and average life expectancy in the realization of a healthy society It is a challenge to build a society with a long and healthy life expectancy through comprehensive prevention and management of lifestyle-related diseases, as well as the elucidation of the factors that explain sex differences in healthy life expectancy, based on the recognition that lifestyle-related diseases in midlife are risk factors for frailty and dementia in old age. Challenges in medical care for building a super-aged and healthy society The challenges include promoting clinical guidelines suitable for older people, including lifestyle-related disease management, promoting comprehensive research on aging (basic research, clinical research and community collaboration research), and embodying a paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care." Furthermore, the key to the future of integrated community care is the development of a comprehensive medical care system for older people in each region and the development of the next generation of medical personnel. Dissemination of frailty prevention measures in a super-aged society The concept of frailty encompasses the meaning of multifacetedness and reversibility; therefore, a comprehensive approach is required, including the renewal of conventional prevention activities in each region, such as the nutritional status of older people, physical activity including exercise, and various opportunities for social participation and participation conditions. Challenges of an unstable diet and undernutrition in older people According to the National Health and Nutrition Examination Survey of Japan, energy and protein intakes are low in Japanese people aged ≥75 years; particularly in people aged ≥80 years, low and insufficient intake of nutrients are prominent. Undernutrition in older people is increasing and is more pronounced in women. There are multiple factors behind this, including social factors, such as living alone, eating alone, poverty and other social factors, as well as problems with access to food security. Pharmacotherapy for older people: measures against polypharmacy In addition to the problems of adverse drug events, drug interactions, duplication of effects and the presence of drugs that "require particularly careful administration," it is also necessary to take measures against polypharmacy in older people, as well as medical economic issues, such as high drug costs and large amounts of remaining drugs. Barriers to this measure include multiple medical institution visits for each disease, lack of coordination between professions, and lack of understanding by patients and families. Role of local communities in a healthy society The decline in the working-age population is also a major challenge; however, we need to make a shift to use this declining birthrate and aging population as an opportunity rather than a crisis. As we look ahead to the coming of the 100-year age of life, we rethink the creation of a comprehensive society and community, and aim to create an age-free society where everyone can play an active role and live in peace, regardless of age. CONTENTS OF THE PROPOSAL: In this report, we have put together a vision for the future of an aging Japanese society from a broader perspective of how the environment and local communities should be, rather than simply from the perspective of individual health. We aim to convey this proposal to the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, the Cabinet Office, and various professional organizations. The paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care" should be promoted for the development of a healthy society While further promoting pre-emptive medical care in the medical care for older people, the development of multidisciplinary medical guidelines appropriate for older people should be promoted at the same time. In addition, we should promote basic aging research, clinical research (including the long-term care field) and transitional research that cover regional areas. Furthermore, while promoting the paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care," the development of various comprehensive medical treatment systems for older people and the strengthening of integrated community care systems should be promoted. Development of the next generation of medical personnel to comprehensively deal with geriatric care, including training geriatric specialists, should be promoted As the number of older people with multimorbidities and frailty rapidly increases in the future, we should promote the development of the next generation of medical personnel who can comprehensively handle medical care for older people, including training leading geriatricians in cooperation with multiple professions in the integrated community care system to provide sufficient medical care. Countermeasures for frailty in older people should be promoted from medical and community planning perspectives To address frailty, which requires comprehensive evaluation and intervention, the three pillars of frailty prevention (nutrition, exercise and social participation) should be incorporated and addressed as part of community development within each municipality, taking into account local characteristics. In particular, it is necessary to revise the way of thinking about nutrition management in older people and the guidelines of the societies in the field. In addition, it is important to strengthen industry-academia-government-private partnerships in each region, taking into account not only medical issues, but also social factors, and encourage the development of momentum in the entire region regarding measures against undernutrition in older people. Polypharmacy measures should be promoted in pharmacotherapy for older people It is necessary to promote cooperation between physicians and pharmacists, establish other multiprofessional cooperation systems, and develop medical and long-term care insurance systems to support this. It is also essential to change the public's mindset, and awareness-raising activities at all levels are required, including the enhancement of educational materials for medical caregivers and the general public. In addition, the economic impact of healthcare using big data should be timely clarified. Innovation in medical and urban planning perspectives should be promoted In the future, it will be necessary to modify and update multidisciplinary approaches such as social participation (e.g. participation in a salon) with a view to innovation in both medical care and community development, especially on the idea of a symbiotic community. In addition, industry-academia-government-private partnership is necessary, including all aforementioned, such as places where people can play an active role in the rest of their lives (such as employment), promotion of human connections, promotion of technology to support older people and support for daily life. Geriatr Gerontol Int 2021; 21: 601-613.

Importance of lymph node immune responses in MSI-H/dMMR colorectal cancer.

Patients with colorectal cancers (CRCs) generally exhibit improved survival through intensive lymph node (LN) dissection. However, recent progress in cancer immunotherapy revisits the potential importance of regional LNs, where T cells are primed to attack tumor cells. To elucidate the role of regional LN, we investigated the immunological status of nonmetastatic regional LN lymphocytes (LNLs) in comparison with those of the tumor microenvironment (tumor-infiltrating lymphocytes; TILs) using flow cytometry and next-generation sequencing. LNLs comprised an intermediate level of the effector T cell population between peripheral blood lymphocytes (PBLs) and TILs. Significant overlap of the T cell receptor (TCR) repertoire was observed in microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) CRCs with high tumor mutation burden (TMB), although limited TCRs were shared between nonmetastatic LNs and primary tumors in microsatellite stable/MMR proficient (MSS/pMMR) CRC patients with low TMB. In line with the overlap of the TCR repertoire, an excessive LN dissection did not provide a positive impact on long-term prognosis in our MSI-H/dMMR CRC cohort (n = 130). We propose that regional LNs play an important role in antitumor immunity, particularly in MSI-H/dMMR CRCs with high TMB, requiring care to be taken regarding excessive nonmetastatic LN dissection in MSI-H/dMMR CRC patients.

Palladium-Induced Temporal Internalization of MHC Class I Contributes to T Cell-Mediated Antigenicity.

Palladium (Pd) is a widely used metal and extremely important biomaterial for the reconstruction of occlusions during dental restorations. However, metallic biomaterials can cause serious allergic reactions, such as Pd-related oral mucositis seen in dentistry. Metal allergy is categorized as a type IV allergy and we demonstrated that CD8 T cells play an important role in Pd allergy previously. As TCR of CD8 T cells recognizes MHC class I/peptide complex, the antigen specificity to this complex seems to be generated during Pd allergy. However, it remains unknown if Pd affects the MHC class I/peptide complex. In this study, we investigated the behavior of the MHC class I/peptide complex in response to Pd treatment. We found that PdCl2 treatment altered peptide presentation on MHC class I and that co-culture with Pd-treated DC2.4 cells induced activation of Pd-responsive TCR-expressing T cell line. Furthermore, PdCl2 treatment induced temporal MHC class I internalization and inhibition of membrane movement suppressed Pd-induced T cell-mediated antigenicity. These data suggest that Pd-induced MHC class I internalization is critical for generation of antigenicity through a mechanism including differential peptide loading on MHC class I, which results in Pd allergy.

Point mutation bias in SARS-CoV-2 variants results in increased ability to stimulate inflammatory responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces severe pneumonia and is the cause of a worldwide pandemic. Coronaviruses, including SARS-CoV-2, have RNA proofreading enzymes in their genomes, resulting in fewer gene mutations than other RNA viruses. Nevertheless, variants of SARS-CoV-2 exist and may induce different symptoms; however, the factors and the impacts of these mutations are not well understood. We found that there is a bias to the mutations occurring in SARS-CoV-2 variants, with disproportionate mutation to uracil (U). These point mutations to U are mainly derived from cytosine (C), which is consistent with the substrate specificity of host RNA editing enzymes, APOBECs. We also found the point mutations which are consistent with other RNA editing enzymes, ADARs. For the C-to-U mutations, the context of the upstream uracil and downstream guanine from mutated position was found to be most prevalent. Further, the degree of increase of U in SARS-CoV-2 variants correlates with enhanced production of cytokines, such as TNF-α and IL-6, in cell lines when compared with stimulation by the ssRNA sequence of the isolated virus in Wuhan. Therefore, RNA editing is a factor for mutation bias in SARS-CoV-2 variants, which affects host inflammatory cytokines production.

Nickel ions bind to HSP90ß and enhance HIF-1α-mediated IL-8 expression.

Nickel ions (Ni2+) eluted from biomedical devices cause inflammation and Ni allergy. Although Ni2+ and Co2+ elicit common effects, Ni2+ induces a generally stronger inflammatory reaction. However, the molecular mechanism by which Ni2+ and Co2+ induce such different responses remains to be elucidated. In the present study, we compared the effects of Ni2+ and Co2+ on the expression of interleukin (IL)-8 in human monocyte THP-1 cells. We report that NiCl2 but not CoCl2 induced the expression of IL-8; in contrast, CoCl2 elicited a higher expression of hypoxia-inducible factor-1α (HIF-1α). The NiCl2-induced expression of IL-8 in late phase was blocked by a HIF-1α inhibitor, PX-478, indicating that NiCl2 targets additional factors responsible for activating HIF-1α. To identify such targets, proteins that bound preferentially to Ni-NTA beads were analyzed by LC/MS/MS. The analysis yielded heat shock protein 90ß (HSP90ß) as a possible candidate. Furthermore, Ni2+ reduced the interaction of HSP90ß with HIF-1α, and instead promoted the interaction between HIF-1α and HIF-1ß, as well as the nuclear localization of HIF-1α. Using various deletion variants, we showed that Ni2+ could bind to the linker domain on HSP90ß. These results suggest that HSP90ß plays important roles in Ni2+-induced production of IL-8 and could be a potential target for the regulation of Ni2+-induced inflammation.

IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting.

Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution.

Quantitative in vivo biocompatibility of new ultralow-nickel cobalt-chromium-molybdenum alloys.

Nickel (Ni) eluted from metallic biomaterials is widely accepted as a major cause of allergies and inflammation. To improve the safety of cobalt-chromium-molybdenum (Co-Cr-Mo) alloy implants, new ultralow-Ni Co-Cr-Mo alloys with and without zirconium (Zr) have been developed, with Ni contents of less than 0.01%. In the present study, we investigated the biocompatibility of these new alloys in vivo by subcutaneously implanting pure Ni, conventional Co-Cr-Mo, ultralow-Ni Co-Cr-Mo, and ultralow-Ni Co-Cr-Mo with Zr wires into the dorsal sides of mice. After 3 and 7 days, tissues around the wire were excised, and inflammation; the expression of IL-1ß, IL-6, and TNF-α; and Ni, Co, Cr, and Mo ion release were analyzed using histological analyses, qRT-PCR, and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Significantly larger amounts of Ni eluted from pure Ni wires than from the other wires, and the degree of inflammation depended on the amount of eluted Ni. Although no significant differences in inflammatory reactions were identified among new alloys and conventional Co-Cr-Mo alloys in histological and qRT-PCR analyses, ICP-MS analysis revealed that Ni ion elution from ultralow-Ni Co-Cr-Mo alloys with and without Zr was significantly lower than from conventional Co-Cr-Mo alloys. Our study, suggests that the present ultralow-Ni Co-Cr-Mo alloys with and without Zr have greater safety and utility than conventional Co-Cr-Mo alloys. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1505-1513, 2016.

Activation of the NLRP3 inflammasome by cellular labile iron.

Cellular labile iron, which contains chelatable redox-active Fe(2+), has been implicated in iron-mediated cellular toxicity leading to multiple organ dysfunction. Iron homeostasis is controlled by monocytes/macrophages through their iron recycling and storage capacities. Furthermore, iron sequestration by monocytes/macrophages is regulated by pro-inflammatory cytokines including interleukin-1, highlighting the importance of these cells in the crosstalk between inflammation and iron homeostasis. However, a role for cellular labile iron in monocyte/macrophage-mediated inflammatory responses has not been defined. Here we describe how cellular labile iron activates the NLRP3 inflammasome in human monocytes. Stimulation of lipopolysaccharide-primed peripheral blood mononuclear cells with ferric ammonium citrate increases the level of cellular Fe(2+) levels in monocytes and induces production of interleukin-1ß in a dose-dependent manner. This ferric ammonium citrate-induced interleukin-1ß production is dependent on caspase-1 and is significantly inhibited by an Fe(2+)-specific chelator. Ferric ammonium citrate consistently induced interleukin-1ß secretion in THP1 cells, but not in NLRP3-deficient THP1 cells, indicating a requirement for the NLRP3 inflammasome. Additionally, activation of the inflammasome is mediated by potassium efflux, reactive oxygen species-mediated mitochondrial dysfunction, and lysosomal membrane permeabilization. Thus, these results suggest that monocytes/macrophages not only sequestrate iron during inflammation, but also mediate inflammation in response to cellular labile iron, which provides novel insights into the role of iron in chronic inflammation.

NKG2D functions as an activating receptor on natural killer cells in the common marmoset (Callithrix jacchus).

The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.

Effect of silica particle size on macrophage inflammatory responses.

Amorphous silica particles, such as nanoparticles (<100 nm diameter particles), are used in a wide variety of products, including pharmaceuticals, paints, cosmetics, and food. Nevertheless, the immunotoxicity of these particles and the relationship between silica particle size and pro-inflammatory activity are not fully understood. In this study, we addressed the relationship between the size of amorphous silica (particle dose, diameter, number, and surface area) and the inflammatory activity (macrophage phagocytosis, inflammasome activation, IL-1ß secretion, cell death and lung inflammation). Irrespective of diameter size, silica particles were efficiently internalized by mouse bone marrow-derived macrophages via an actin cytoskeleton-dependent pathway, and induced caspase-1, but not caspase-11, activation. Of note, 30 nm-1000 nm diameter silica particles induced lysosomal destabilization, cell death, and IL-1ß secretion at markedly higher levels than did 3000 nm-10000 nm silica particles. Consistent with in vitro results, intra-tracheal administration of 30 nm silica particles into mice caused more severe lung inflammation than that of 3000 nm silica particles, as assessed by measurement of pro-inflammatory cytokines and neutrophil infiltration in bronchoalveolar lavage fluid of mice, and by the micro-computed tomography analysis. Taken together, these results suggest that silica particle size impacts immune responses, with submicron amorphous silica particles inducing higher inflammatory responses than silica particles over 1000 nm in size, which is ascribed not only to their ability to induce caspase-1 activation but also to their cytotoxicity.

Characterization of T cell receptors of Th1 cells infiltrating inflamed skin of a novel murine model of palladium-induced metal allergy.

Metal allergy is categorized as a delayed-type hypersensitivity reaction, and is characterized by the recruitment of lymphocytes into sites of allergic inflammation. Because of the unavailability of suitable animal models for metal allergy, the role of T cells in the pathogenesis of metal allergy has not been explored. Thus, we developed a novel mouse model for metal allergy associated with infiltration of T cells by multiple injections of palladium (Pd) plus lipopolysaccharide into the footpad. Using this model, we characterized footpad-infiltrating T cells in terms of phenotypic markers, T cell receptor (TCR) repertoires and cytokine expression. CD3+ CD4+ T cells accumulated in the allergic footpads 7 days after Pd challenge. The expression levels of CD25, interleukin-2, interferon-γ and tumor necrosis factor, but not interleukin-4 and interleukin-5, increased in the footpads after challenge, suggesting CD4+ T helper 1 (Th1) cells locally expanded in response to Pd. Infiltrated T cells in the footpads frequently expressed AV18-1 and BV8-2 T cell receptor (TCR) chains compared with T cells in the lymph nodes and exhibited oligoclonality. T-cell clones identified from Pd-allergic mouse footpads shared identical CDR3 sequences containing AV18-1 and BV8-2. These results suggest that TCR AV18-1 and BV8-2 play dominant and critical parts in the antigen specificity of Pd-specific Th1 cells.

Accumulation of invariant NKT cells into inflamed skin in a novel murine model of nickel allergy.

Nickel (Ni) can cause delayed-type hypersensitivity reactions, which are thought to be mediated by the accumulation of T cells into inflamed skin. Accumulated T cells at the developmental stages in metal allergy are poorly characterized because a suitable animal model has not been established. To investigate the accumulated T cells in allergic inflamed skin, we generated a novel murine model of Ni-induced allergy. The murine model of Ni allergy was induced by two sensitizations of Ni plus lipopolysaccharide solution into the groin followed by three challenges with Ni solution into the footpad. Here we show that a specific TCR repertoire bearing Vα14Jα18, called natural killer (NK) T cells, was expanded monoclonally in BALB/c or C57BL/6 mice. Accumulation of NKT cells was characterized as CD4(+) or CD4(-)CD8(-) T cells. These results suggested that NKT cells are major pathogenic T cells at the elicitation phase of Ni allergy.

Fratricide of natural killer cells dressed with tumor-derived NKG2D ligand.

The natural killer group 2 membrane D (NKG2D) activating receptor plays crucial roles not only in host defense against tumors and viral infections, but also in autoimmune diseases. After NKG2D-mediated activation, Natural killer (NK) cells must be regulated to avoid potentially harmful reactivity. However, the negative regulation of these activated NK cells is poorly understood. Here, we reveal that the engagement of NKG2D by its ligand elicits not only target cell lysis, but also NK cell fratricide. Conventional mouse NK cells underwent cell death when cocultured with RMA cells expressing the NKG2D ligand retinoic acid early-inducible protein 1 (Rae-1), but not with RMA cells lacking MHC class I. NK cells from mice deficient for DAP10 and DAP12 or perforin did not undergo death, highlighting the importance of the NKG2D pathway for NK cell death. However, NKG2D does not transmit direct death signals in NK cells. Rather, the interaction between NKG2D and Rae-1 allowed NK cells to acquire tumor-derived Rae-1 by a membrane transfer process known as "trogocytosis," which was associated with clathrin-dependent NKG2D endocytosis. NK cells dressed with Rae-1 were lysed by neighboring NK cells through the NKG2D-induced perforin pathway in vitro and in vivo. These results provide the unique NKG2D function in negative regulation of activated NK cells.

NK-cell fratricide: Dynamic crosstalk between NK and cancer cells.

The intercellular transfer of plasma membrane patches, also known as trogocytosis, has a strong impact on the function and fate of immune cells. We have recently shown that natural killer (NK) cells undergo fratricide following the trogocytosis-mediated acquisition of tumor-derived NKG2D ligands. Malignant cells may therefore employ trogocytosis to escape NKG2D-mediated immune responses.

IFN-γ production by lung NK cells is critical for the natural resistance to pulmonary metastasis of B16 melanoma in mice.

NK cells are effector lymphocytes playing a critical role in the natural resistance against tumors. However, the precise mechanisms underlying NK cell-mediated natural resistance against tumor metastasis are still unrevealed. B16 cells, mouse melanoma cells, were resistant to freshly isolated NK cell-mediated killing; nevertheless, NK cells were critical for natural resistance against experimental lung metastasis of B16 cells. We found that lung metastasis was increased significantly in IFN-γ(-/-) mice but not pfp(-/-), IFN-αR(-/-), or IL-12/IL-18(-/-) mice. Interestingly, freshly isolated lung NK cells, but not spleen or liver NK cells, displayed augmented IFN-γ production after B16 inoculation. Adoptive transfer of pfp(-/-) NK cells, but not IFN-γ(-/-) NK cells, significantly decreased B16 lung metastasis in IFN-γ(-/-) and pfp/IFN-γ(-/-)mice. Lung metastases of IFN-γRDN B16 was also increased in NK cell-depleted or IFN-γ(-/-) mice, suggesting that the IFN-γ response of host cells was required in the NK cell and IFN-γ-mediated antimetastatic effect. Our results demonstrate that IFN-γ production from lung resident NK cells is a key response in the natural resistance to the experimental lung metastasis of NK cell-resistant tumor cells.

The ECS(SPSB) E3 ubiquitin ligase is the master regulator of the lifetime of inducible nitric-oxide synthase.

The ubiquitin-proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C-Cullin-5-SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70-interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime.