Miyuu Tanaka

M1- and M2-macrophage polarization in rat liver cirrhosis induced by thioacetamide (TAA), focusing on Iba1 and galectin-3

INTRODUCTION Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1(+) (Iba1(+)) and galectin-3(+) (Gal-3(+)) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1(+) and Gal-3(+) macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1(+) and Gal-3(+) macrophages in chemical-induced liver cirrhosis. MATERIALS AND METHODS Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs. RESULTS Activation of myofibroblasts was most pronounced in livers at week 15. CD68(+) (M1), CD204(+) (M2), Iba1(+) and Gal-3(+) macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1β, IL-6, and TNF-α) and M2-(TGF-β1, IL-4, and IL-10) related factors. Iba1(+) and Gal-3(+) macrophages showed both M1- and M2-immunophenotypes. CD163(+) macrophages showed a persistent increase, consistent with TGF-β1 upregulation. MHC class II(+) macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis. CONCLUSION Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1(+) and Gal-3(+) macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.

The F Gene of the Osaka-2 Strain of Measles Virus Derived from a Case of Subacute Sclerosing Panencephalitis Is a Major Determinant of Neurovirulence

ABSTRACT Measles virus (MV) is the causative agent for acute measles and subacute sclerosing panencephalitis (SSPE). Although numerous mutations have been found in the MV genome of SSPE strains, the mutations responsible for the neurovirulence have not been determined. We previously reported that the SSPE Osaka-2 strain but not the wild-type strains of MV induced acute encephalopathy when they were inoculated intracerebrally into 3-week-old hamsters. The recombinant MV system was adapted for the current study to identify the gene(s) responsible for neurovirulence in our hamster model. Recombinant viruses that contained envelope-associated genes from the Osaka-2 strain were generated on the IC323 wild-type MV background. The recombinant virus containing the M gene alone did not induce neurological disease, whereas the H gene partially contributed to neurovirulence. In sharp contrast, the recombinant virus containing the F gene alone induced lethal encephalopathy. This phenotype was related to the ability of the F protein to induce syncytium formation in Vero cells. Further study indicated that a single T461I substitution in the F protein was sufficient to transform the nonneuropathogenic wild-type MV into a lethal virus for hamsters.

Generation of Functional Platelets from Canine Induced Pluripotent Stem Cells

Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.

Immunohistochemical characterization of glial fibrillary acidic protein (GFAP)-expressing cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

Hepatic stellate cells, the principal fibrogenic cell type in the liver, are known to express the astrocyte marker glial fibrillary acidic protein (GFAP). However, the exact role of GFAP-expressing cells in liver fibrosis remains to be elucidated. In this study, cellular properties of GFAP-expressing cells were investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100 mg/kg BW, twice a week) and examined at post first injection weeks 5, 10, 15, 20 and 25. Appearance of GFAP-expressing myofibroblasts peaked at week 15, associated with fibrosis progression. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and alpha-smooth muscle actin. Some GFAP-positive myofibroblasts co-expressed nestin (neural stem cell marker), while a few co-expressed A3 (mesenchymal stem cell marker) and Thy-1 (immature mesenchymal cell marker). A few GFAP expressing cells underwent both mitosis and apoptosis. These results indicate that there is a dynamic participation of GFAP-expressing myofibroblasts in rat liver cirrhosis, and that they are mainly derived from hepatic stellate cells, and partly from cells in the stem cell lineage. These findings, which were shown for the first time in detail, would be useful to understand the role of GFAP-expressing myofibroblasts in the pathogenesis of chemically induced liver cirrhosis.

Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury

Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1-10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.

Slowly progressive cholangiofibrosis induced in rats by α-naphthylisothiocyanate (ANIT), with particular references to characteristics of macrophages and myofibroblasts.

A progressive cholangiofibrosis was developed as an animal model in 6-week-old male F344 rats by repeated intraperitoneal injections of α-naphthylisothiocyanate (ANIT) for 19 weeks; liver samples were examined at post-first injection (PFI) weeks 3, 7, 10, 13, 16 and 19, focusing on characteristics of macrophages and myofibroblasts by immunohistochemical analyses. In the affected Glissons sheath consisting of inflammatory cell infiltrates, bile duct proliferation and advancing fibrosis, the number of macrophages reacting to OX6 (recognizing MHC class II) increased consistently (PFI weeks 3-19), suggesting a central role of antigen presenting cells in the biliary fibrosis; macrophages reacting to ED1 (CD68, reflecting phagocytic activity) and ED2 (CD163, relating to proinflammatory factor production) showed a significantly increased number at PFI weeks 7-19 and PFI weeks 13-19, respectively. Interestingly, macrophages positive for SRA-E5 (CD204, reflecting lipid metabolism) increased at PFI weeks 7-19, and the appearance was limited in the sinusoids around the affected Glissons sheath. Myofibroblasts appearing in the affected Glissons sheath reacted to vimentin and desmin at early (PFI weeks 3-7) and mid (PFI weeks 10-13) stages, and then they came to strongly express α-smooth muscle actin at late stage (PFI weeks 16-19). This study shows that macrophages exhibit heterogeneous properties depending on stages and locations; in association with such macrophage populations, myofibroblasts expressing various cytoskeletons participate in cholangiofibrosis. These characteristics would be useful in evaluating the pathogenesis of possible cholangio-toxicants.

Two Cases of Lacaziosis in Bottlenose Dolphins (Tursiops truncatus) in Japan

Lacaziosis, formerly called lobomycosis, caused by Lacazia loboi, is a zoonotic mycosis found in humans and dolphins and is endemic in the countries on the Atlantic Ocean. Although the Japanese coast is not considered an endemic area, photographic records of lacaziosis-like skin lesions were found in bottlenose dolphins (Tursiops truncatus) that were migrating in the Goto Islands (Nagasaki Prefecture, Japan). We diagnosed 2 cases of lacaziosis in bottlenose dolphins captured simultaneously at the same coast within Japanese territory on the basis of clinical characteristics, cytology, histopathology, immunological tests, and detection of partial sequences of a 43 kDa glycoprotein coding gene (gp43) with a nested-PCR system. The granulomatous skin lesions from the present cases were similar to those found in animals from endemic areas, containing multiple budding and chains of round yeast cells and positive in the immune-staining with anti-Paracoccidioides brasiliensis serum which is a fungal species related to L. loboi; however, the gp43 gene sequences derived from the present cases showed 94.1% homology to P. brasiliensis and 84.1% to L. loboi. We confirmed that the causative agent at the present cases was different genotype of L. loboi from Amazon area.

Expressions of Iba1 and galectin-3 (Gal-3) in thioacetamide (TAA)-induced acute rat liver lesions.

Ionized calcium binding adaptor molecule 1 (Iba1) is associated with membrane ruffling and motility of cells. Galectin-3 (Gal-3) is a β-galactoside binding animal lectin, and regulates fibrogenesis probably through transforming growth factor-β1. To evaluate macrophage properties, expressions of Iba1 and Gal-3 were investigated, in relation to macrophages expressing CD68 (ED1; reflecting increased phagocytosis) and CD163 (ED2; implying proinflammatory factor productions) in centrilobular lesions induced in rat livers with thioacetamide (TAA; 300 mg/kg body weight, once intraperitoneally). In agreement with expression patterns of CD68(+) and CD163(+) macrophages, cells reacting to Iba1 and Gal-3 were increased in numbers on post-injection (PI) days 1-5, peaking on day 2; thereafter, the positive cells gradually decreased to control levels until PI days 7 and 10. The increased expressions of Iba1 and Gal-3 were confirmed at mRNA levels by the RT-PCR. Double immunofluorescence staining on PI days 2 and 3 demonstrated Iba1 expression in 15-46% of CD68(+) and CD163(+) macrophages, and Gal-3 expression in 65-82% of CD68(+) and CD163(+) macrophages; Gal-3 expression was observed in 84-93% of Iba1(+) cells. Interestingly, Gal-3 was also expressed in a small number of α-smooth muscle actin-positive myofibroblasts in fibrotic lesions developed in injured centrilobular areas. These findings indicate that macrophages with various functions can participate in development of liver lesions and resultant fibrosis. Besides CD68 and CD163, Iba1 and Gal-3 immunohistochemistry for macrophages would be useful to analyze the pathogenesis behind developing hepatotoxicity.

Immunophenotypical analysis of myofibroblasts and mesenchymal cells in the bleomycin-induced rat scleroderma, with particular reference to their origin.

Cellular characteristics of myofibroblasts and its possible origin with mesenchymal stem cell nature in scleroderma remain to be investigated. We analyzed these cells in scleroderma induced in F344 rats by bleomycin (BLM) by immunolabeling using a panel of marker antibodies for cytoskeletons (vimentin, desmin, α-smooth muscle actin (α-SMA)) and stromal stem cells (Thy-1, A3). Skin samples were collected at 1, 2, 3, and 4 weeks after initiation of subcutaneous injections of BLM (100 μl of 1 mg/ml, daily). In double immunofluorescence, myofibroblasts reacting simultaneously to α-SMA, vimentin, and Thy-1 were seen in sclerotic lesions with a time-dependent increase. Mesenchymal cells in the perifollicular dermal sheath (PDS) displayed increased reactivity for Thy-1 and vimentin, but α-SMA expression did not increase in these cells. In double immunofluorescence, both myofibroblasts and pericytes in newly formed blood vessels in sclerotic lesions co-expressed α-SMA, vimentin and Thy-1, and the PDS cells and pericytes reacted simultaneously to A3, Thy-1 and vimentin. Desmin-positive cells were infrequently seen around the blood vessels. Based on these findings, the PDS cells and pericytes may be involved as possible progenitors of myofibroblasts in sclerotic lesions in the stromal stem cell lineage. Interestingly, increased number of TUNEL-positive apoptotic epithelial cells in the atrophied hair follicles significantly correlated with increase in immunohistochemical scoring of vimentin and Thy-1 in the PDS. Apoptosis in the hair follicle might have mediate the perifollicular fibrosis, resulting in extensive scleroderma. The present findings would provide new insights in the pathogenesis of BLM-induced scleroderma in terms of myofibroblasts and its origin.

M1-/M2-macrophage polarization in pseudolobules consisting of adipohilin-rich hepatocytes in thioacetamide (TAA)-induced rat hepatic cirrhosis

INTRODUCTION Liver steatosis is the most frequent liver disease and may further develop into non-alcoholic steatohepatitis (NASH), liver cirrhosis, and finally hepatocellular carcinoma. Adipophilin (Adp) is localized on lipid droplet membrane in cytoplasm, and its increased expression is related to development of steatosis and NASH. The relationship between M1-/M2-macrophage polarization and Adp-rich hepatocyte-consisting pseudolobules (PLs) was investigated in thioacetamide (TAA)-induced rat cirrhosis. MATERIALS AND METHOD F344 rats were injected twice weekly with TAA (100mg/kg bodyweight) and sacrificed at post-first injection (PFI) weeks 5, 10, 15, 20, 25 and 32. Macrophage immunophenotypes and Adp-containing hepatocytes were analyzed by single immunolabeling. Adp and M1-/M2-related factors were analyzed by real -time RT-PCR. RESULTS PLs consisting exclusively of Adp-containing hepatocytes (Adp-positive) and PLs consisting of few Adp-containing hepatocytes (Adp-negative) were clearly distinguishable at PFI week 20 onwards. The numbers of M1-macrophages (reacting to CD68 and Iba1) and M2- macrophages (reacting to CD163, CD204 and Gal-3) were considerably greater in Adp-positive PLs. Expressions for both M1 (TNF-α, MCP-1, and Iba1)- and M2 (IL-4, TGF-β1, Gal-3, and Hsp25)-related factors were markedly higher in Adp-positive PLs at PFI week 25. Interestingly, MHC class II-positive macrophages/dendritic cells were increased in Adp-positive clusters/foci at the early stages at PFI weeks 5 and 10, and the level was gradually decreased thereafter. CONCLUSIONS M1-/M2-macrophages may simultaneously participate in the pathogenesis of steatosis in TAA-induced cirrhosis through M1- and M2-related factors. MHC class II cells may be responsible for steatosis at early stages, suggesting different functions from the above M1-/M2-macropahges.