Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C−and CD11c− /Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C− cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c−/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH

Low-intensity pulsed ultrasound induces apoptosis in osteoclasts: Fish scales are a suitable model for the analysis of bone metabolism by ultrasound

Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3 h of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal-less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18 h of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in osteoclasts using the TUNEL (TdT-mediated dUTP nick end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3 h of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure. © 2016 Elsevier Inc.Embargo Period 12 month

DNA Double-Strand Breaks Induced by Cavitational Mechanical Effects of Ultrasound in Cancer Cell Lines

Ultrasonic technologies pervade the medical field: as a long established imaging modality in clinical diagnostics; and, with the emergence of targeted high intensity focused ultrasound, as a means of thermally ablating tumours. In parallel, the potential of [non-thermal] intermediate intensity ultrasound as a minimally invasive therapy is also being rigorously assessed. Here, induction of apoptosis in cancer cells has been observed, although definitive identification of the underlying mechanism has thus far remained elusive. A likely candidate process has been suggested to involve sonochemical activity, where reactive oxygen species (ROS) mediate the generation of DNA single-strand breaks. Here however, we provide compelling new evidence that strongly supports a purely mechanical mechanism. Moreover, by a combination of specific assays (neutral comet tail and staining for γH2AX foci formation) we demonstrate for the first time that US exposure at even moderate intensities exhibits genotoxic potential, through its facility to generate DNA damage across multiple cancer lines. Notably, colocalization assays highlight that ionizing radiation and ultrasound have distinctly different signatures to their respective γH2AX foci formation patterns, likely reflecting the different stress distributions that initiated damage formation. Furthermore, parallel immuno-blotting suggests that DNA-PKcs have a preferential role in the repair of ultrasound-induced damage

Parathyroid hormone 1 (1-34) acts on the scales and involves calcium metabolism in goldfish

金沢大学環日本海域環境研究センターThe effect of fugu parathyroid hormone 1 (fugu PTH1) on osteoblasts and osteoclasts in teleosts was examined with an assay system using teleost scale and the following markers: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Synthetic fugu PTH1 (1-34) (100 pg/ml-10 ng/ml) significantly increased ALP activity at 6 h of incubation. High-dose (10 ng/ml) fugu PTH1 significantly increased ALP activity even after 18 h of incubation. In the case of TRAP activity, fugu PTH1 did not change at 6 h of incubation, but fugu PTH1 (100 pg/ml-10 ng/ml) significantly increased TRAP activity at 18 h. Similar results were obtained for human PTH (1-34), but there was an even greater response with fugu PTH1 than with human PTH. In vitro, we demonstrated that both the receptor activator of the NF-κB ligand in osteoblasts and the receptor activator NF-κB mRNA expression in osteoclasts increased significantly by fugu PTH1 treatment. In an in vivo experiment, fugu PTH1 induced hypercalcemia resulted from the increase of both osteoblastic and osteoclastic activities in the scale as well as the decrease of scale calcium contents after fugu PTH1 injection. In addition, an in vitro experiment with intramuscular autotransplanted scale indicated that the ratio of multinucleated osteoclasts/mononucleated osteoclasts in PTH-treated scales was significantly higher than that in the control scales. Thus, we concluded that PTH acts on osteoblasts and osteoclasts in the scales and regulates calcium metabolism in goldfish. © 2011 Elsevier Inc. All rights reserved

Pitfalls in global normalization of ChIP-seq data in CD4+ T cells treated with butyrate: A possible solution strategy

Regulatory T cells (Treg) play a central role in the suppression of inflammatory and allergic responses. Colonization of certain gut commensal microbes such as Clostridia class IV and XIVa in the gut can induce development of colonic Treg cells contributing to the maintenance of gut immune homeostasis. Clostridia-derived butyrate promotes the differentiation of naïve T cells into Treg cells through upregulation of Foxp3, the master transcription factor of Treg cells. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that treatment of naïve T cells with butyrate induces Treg-polarizing conditions by enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus. In general, global normalization was utilized for ChIP-seq analysis to compare the data obtained from two or more samples. However, global normalization is not appropriate for the evaluation of ChIP-seq data when treatment can affect the total amount of target protein. Here, we introduce a unique normalization method for ChIP-seq analysis in cells treated with butyrate, a pan-HDAC inhibitor that is likely to affect total acetylation levels of histone H3

Alkannin, HSP70 inducer, protects against UVB-induced apoptosis in human keratinocytes.

Alkannin is an active constituent from the root extract of Alkanna tinctoria of the Boraginaceae family and it may have utility as a heat shock protein 70 (HSP70) inducer in living organisms. Here, the effects of alkannin-induced HSP70 on ultraviolet (UV) B (40 mJ/cm(2))-induced apoptosis were investigated in human keratinocyte HaCaT cells. Pretreatment of cells with alkannin (1 µM) caused significant inhibition of UVB-induced apoptosis and caspase-3 cleavage. On the other hand, the addition of KNK437 (HSP70 inhibitor) reversed the action of alkannin increasing UVB-induced apoptosis in a dose-dependent manner. In addition, differences in gene expression associated with the suppression of UVB-induced apoptosis in the presence of alkannin were investigated using Gene Chip assay. Our results indicate that alkannin suppresses UVB-induced apoptosis through the induction of HSP70 in human keratinocytes, and therefore, we suggest the usefulness of using alkannin as an antiaging agent

Effect of Wheat-Derived Arabinoxylan on the Gut Microbiota Composition and Colonic Regulatory T Cells

The health benefits of wheat-derived arabinoxylan, a commonly consumed dietary fiber, have been studied for decades. However, its effect on the gut microenvironment and inflammatory bowel disease remains unclear. The objective of this study was to understand the effect of wheat-derived arabinoxylan on gut microbiota, colonic regulatory T cells (Tregs), and experimental colitis. In this study, healthy and chronic colitis model mice were fed chow containing cellulose or wheat-derived arabinoxylan for 2–6 weeks and subjected to subsequent analysis. A 16S-based metagenomic analysis of the fecal DNA revealed that Lachnospiraceae, comprising butyrate-producing and Treg-inducing bacteria, were overrepresented in arabinoxylan-fed mice. In line with the changes in the gut microbiota, both the fecal butyrate concentration and the colonic Treg population were elevated in the arabinoxylan-fed mice. In a T cell transfer model of chronic colitis, wheat-derived arabinoxylan ameliorated body weight loss and colonic tissue inflammation, which may, in part, be mediated by Treg induction. Moreover, wheat-derived arabinoxylan suppressed TNFα production from type 1 helper T cells in this colitis model. In conclusion, wheat-derived arabinoxylans, by altering the gut microenvironment, may be a promising prebiotic for the prevention of colitis

Induction of Immune Suppression in the Chick by an Optimal Dose of an Immunizing Antigen in the Presence of its Specific Maternal Antibody

Prolonged interference or suppression of maternal antibodies of the humoral immune response of newly hatched chicks to active immunization has been documented; however, the immunological mechanisms responsible for such suppression are still unclear. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP or non-specificIgY antibodies were transferred by yolk sac inoculation to newly hatched chicks, and they were immunized with DNP-KLH or rabbit serum albumen (RSA) at 1 and 4 weeks of age. The concentrations of anti-DNP and anti-RSA antibodies in serum samples of these chicks were measured using an enzyme-linked immunosorbent assay (ELISA). The immune responses of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with an appropriate dose of DNP-KLH were suppressed. However, those of the chicks that received the same high dose of maternal non-specific IgY antibodies and were immunized with an appropriate dose of DNP-KLH and those of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with RSA were not suppressed. On the other hand, suppression of anti-DNP antibody production would not be induced if the chicks received a high dose of antigen specific maternal antibodies and were immunized with a high dose of the same antigen. These results revealed that the immune suppressive effect of maternal antibodies on the immune response of the newly hatched chicks was antigen specific and depended mainly on the ratio of antigen/maternal antibody at the time of immunization