Showing papers by "Ken-ichi Isobe published in 2016"

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

Abstract: Author(s): Klionsky, DJ; Abdelmohsen, K; Abe, A; Abedin, MJ; Abeliovich, H; Arozena, AA; Adachi, H; Adams, CM; Adams, PD; Adeli, K; Adhihetty, PJ; Adler, SG; Agam, G; Agarwal, R; Aghi, MK; Agnello, M; Agostinis, P; Aguilar, PV; Aguirre-Ghiso, J; Airoldi, EM; Ait-Si-Ali, S; Akematsu, T; Akporiaye, ET; Al-Rubeai, M; Albaiceta, GM; Albanese, C; Albani, D; Albert, ML; Aldudo, J; Algul, H; Alirezaei, M; Alloza, I; Almasan, A; Almonte-Beceril, M; Alnemri, ES; Alonso, C; Altan-Bonnet, N; Altieri, DC; Alvarez, S; Alvarez-Erviti, L; Alves, S; Amadoro, G; Amano, A; Amantini, C; Ambrosio, S; Amelio, I; Amer, AO; Amessou, M; Amon, A; An, Z; Anania, FA; Andersen, SU; Andley, UP; Andreadi, CK; Andrieu-Abadie, N; Anel, A; Ann, DK; Anoopkumar-Dukie, S; Antonioli, M; Aoki, H; Apostolova, N; Aquila, S; Aquilano, K; Araki, K; Arama, E; Aranda, A; Araya, J; Arcaro, A; Arias, E; Arimoto, H; Ariosa, AR; Armstrong, JL; Arnould, T; Arsov, I; Asanuma, K; Askanas, V; Asselin, E; Atarashi, R; Atherton, SS; Atkin, JD; Attardi, LD; Auberger, P; Auburger, G; Aurelian, L; Autelli, R

Predominant role of msr(D) over mef(A) in macrolide resistance in Streptococcus pyogenes.

Abstract: In Japan, the number of patients with streptococcal toxic shock syndrome is reported to be increasing. mef(A) gene-positive macrolide-resistant emm1 strains are thought to possibly contribute to the rise in the frequency of STSS. Although analyses of macrolide-resistant mechanisms, including mef(A) resistance, have been performed mainly in Streptococcus pneumoniae, the role of this gene in Streptococcus pyogenes has not been completely investigated. Therefore, to the best of our knowledge, we established the first mef(A)-knockout strain using an emm1-type S. pyogenes strain, and tested its susceptibility to erythromycin, clarithromycin and azithromycin. We found that the antimicrobial susceptibilities were almost identical to those of the parental strain. Hence, we established a knockout strain for another gene, msr(D), that is located immediately downstream of mef(A). The macrolide resistances of the resulting strain significantly decreased, and were further altered when both mef(A) and msr(D) were knocked out. The introduction of the msr(D) gene into a macrolide-sensitive strain conferred more resistance than the introduction of the mef(A) gene. The erythromycin susceptibilities of knockout strains were further dissected using two additional emm4- and emm75-type S. pyogenes strains. We found almost identical results for both strains except for the mef(A) knockout emm4 type, whose susceptibility was altered, although the change was less than that for the msr(D) knockout. These results suggest that both mef(A) and msr(D) are involved in macrolide resistance in S. pyogenes, and that the msr(D) gene plays a more predominant role in macrolide resistance than mef(A).

GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation

Abstract: Growth arrest and DNA damage inducible protein 34 (GADD34) is induced by various cellular stresses, such as DNA damage, endoplasmic reticulum stress, and amino-acid deprivation. Although the major roles of GADD34 are regulating ER stress responses and apoptosis, a recent study suggested that GADD34 is linked to innate immune responses. In this report, we investigated the roles of GADD34 in inflammatory responses against bacterial infection. To explore the effects of GADD34 on systemic inflammation in vivo, we employed a lipopolysaccharide (LPS)-induced murine sepsis model and assessed the lethality, serum cytokine levels, and tissue injury in the presence or absence of GADD34. We found that GADD34 deficiency increased the lethality and serum cytokine levels in LPS-induced sepsis. Moreover, GADD34 deficiency enhanced tissue destruction, cell death, and pro-inflammatory cytokine expression in LPS-induced acute liver injury. Pro-inflammatory cytokine production after LPS stimulation is regulated by the Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway. In vitro experiments revealed that GADD34 suppressed pro-inflammatory cytokine production by macrophages through dephosphorylation of IKKβ. In conclusion, GADD34 attenuates LPS-induced sepsis and acute tissue injury through suppressing macrophage activation. Targeting this anti-inflammatory role of GADD34 may be a promising area for the development of therapeutic agents to regulate inflammatory disorders.

Transfusion of CD206+ M2 Macrophages Ameliorates Antibody-Mediated Glomerulonephritis in Mice

Abstract: Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis, depending on the activated phenotype. In this study, we investigated the protective effects of CD206 + M2 macrophages against nephrotoxic serum nephritis in mice. We found that these immunosuppressive macrophages, derived from bone marrow and stimulated with IL-4/IL-13 [CD206 + M2 bone marrow–derived macrophages (M2BMMs)], protected against renal injury, decreased proteinuria, and diminished the infiltration of CD68 + macrophages, neutrophils, and T cells into glomerular tissue. Comparable therapeutic results were obtained with CD206 + M2 cells derived from induced pluripotent stem cells. Notably, CD206 + M2BMMs, which retained an M2 signature, could elicit a switch of M1 to M2 phenotype in co-cultured macrophages. Moreover, these cells were found to induce the production of regulatory T cells in the spleen and renal draining lymph node. Accordingly, mRNA expression of the T helper 1 cytokines tumor necrosis factor-α, interferon-β, interferon-γ, and IL-12 was significantly reduced in kidneys from mice treated with CD206 + M2BMMs. Taken together, the data suggest that CD206 + M2 may have therapeutic potential against antibody-mediated glomerular injury and presents its therapeutic value for the treatment of crescentic nephritis in humans.

Vancomycin-sensitive bacteria trigger development of colitis-associated colon cancer by attracting neutrophils.

Abstract: Inflammatory bowel disease confers an increased risk of developing colitis-associated colon cancer (CAC). During the active colitis or developing tumor stage, commensal bacteria show dynamic translocation. However, whether alteration of the bacterial composition in the gut causes CAC is still unclear. To clarify the effect of commensal bacteria on CAC development, we employed an azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced murine CAC model treated with or without antibiotics. In addition, we analyzed the effects of antibiotics on infiltration of myeloid cells, colonic inflammatory responses and colorectal cancer formation. We found that vancomycin treatment dramatically suppressed tumor development. In addition, AOM/DSS treatment greatly induced the infiltration of Gr-1high/CD11bhigh neutrophils to the colon, which led to the production of tumor necrosis factor α and inducible nitric oxide synthase. Vancomycin treatment suppressed the infiltration of neutrophils induced by AOM/DSS. Moreover, vancomycin treatment greatly reduced the colon injury and DNA damage caused by AOM/DSS-induced NO radicals. Our results indicate that vancomycin-sensitive bacteria induced colon inflammation and DNA damage by attracting neutrophils into damaged colon tissue, thus promoting tumor formation.

GADD34 Promotes Tumor Growth by Inducing Myeloid-derived Suppressor Cells.

Abstract: Background Tumor hypoxia induces the expression of growth arrest and DNA damage-inducible protein (GADD34). However, the role of GADD34 in tumor growth remains unclear. Materials and methods Gadd34 expression was knocked-down through lentivirus-mediated short hairpin RNA (shRNA) in tumor cells, which were subsequently injected subcutaneously into mice. Tumor volumes and myeloid-derived suppressor cells (MDSCs) were monitored. Isolated MDSCs were incubated with tumor supernatant to investigate the impact of GADD34 on cytokine secretion of MDSCs. Results We observed that reduction of GADD34 expression significantly suppressed tumor, and resulted in decreased accumulation of MDSCs and T-cells, and inhibition of GADD34 reduced secretion of vascular epithelial growth factor α and transforming growth factor β by MDSCs. Conclusion These findings provide a promising strategy for targeting GADD34 activity in order to inhibit tumor growth.

Mechanism of immune aging.

Abstract: Age-related decline of immune system not only increases susceptibility of infection, but also delays of wound healing. Although the number of innate immune cells such as neutro- phils and macrophages does not decline by aging, the functions of these cells such as phagocy- tosis, migration toward infection sites decline by aging. Production of inflammatory cytokines by microglia, one of tissue macrophages increases by aging, which causes Alzheimer disease. Human thymic epithelium, which harbor lymphocytes in thymus for development, show a continuous involution from the first year, which replaced by adipose tissue. Age-related involu- tion of thymic epithelium results in the decrease of nalve T cells. Chronic cytomegalovirus infection induces clonally-expanded CD8 T cells and perturbations in the peripheral T cell repertoire.

Murine model of obesity-induced type II diabetes by GADD34 (PPP1R15A) and other phosphatase 1 regulatory subunits deficiency.

Abstract: Obesity-derived metabolic syndrome is one of the most important medical problems in the world. Recently, we have developed murine model of age-dependent obesity and metabolic syndrome including Type II diabetes by using GADD34deficient mice. GADD34 belongs one of phosphatase 1 regulatory subunits named PPP1R15A. Deficiency of glycogen-targeting regulatory subunits (G subunits) has been shown to develop metabolic diseases. Glycogen synthesis is mainly controlled by glycogen synthase (GS) and glycogen phosphorylase (GP). G subunits work to up-regulate glycogen synthesis by activating GS and suppressing GP. Seven G subunits (PPP1R3A to PPP1R3G) works to increase glycogen. PPP1R3A deficient mice became obese by age and showed insulin resistance. Because GADD34 binds several target proteins and PP1c catalytic subunit, GADD34 (PPP1R15A) has several functions in different stimulations by dephosphorylating target proteins. With age GADD34-deficient mice become obese, developing fatty liver followed by liver cirrhosis, hepatocellular carcinoma, and insulin resistance.