Hossain M. Golbar

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.

Immunohistochemical analyses of the kinetics and distribution of macrophages, hepatic stellate cells and bile duct epithelia in the developing rat liver.

Non-parenchymal cells in the liver consist mainly of Kupffer cells, hepatic stellate (HS) cells and cholangiocytes. To establish base-line data and clarify the nature, this study investigated immunohistochemically the kinetics of these cell populations in developing liver of F344 rats. Samples were collected from fetuses on days 18 and 20, neonates on days 1, 4, 8, 15, and 21, and adults at weeks 5-35. ED1 (CD68)-positive macrophages showed highest number as early as fetal day 18, and then decreased gradually until adulthood. The numbers of macrophages reacting to ED2 (CD163), OX6 (MHC II), and SRA-E5 (scavenger receptor A, CD204) increased after birth (early neonates), and ED2- and SRA-E5-positive cell numbers were maintained until adulthood, but OX6-positive cell number decreased at late stages of neonates and adulthood. ED2- and SRA-E5-positive cells appeared along sinusoids, indicating Kupffer cells, whereas OX6-positive cells were limited in Glissons sheath. Vimentin-positive HS cells were seen consistently from fetuses to adulthood. Desmin- and glial fibrillary acidic protein (GFAP)-positive HS cells tended to be seen in fetuses and early stages of neonates. HS cells reacting to α-smooth muscle actin (α-SMA) were not detectable. Cholangiocytes, reacting to cytokeratin 19 and AE1/AE3, began to be seen on fetus day 18 with faint reaction, and interlobular bile ducts were completed in Glissons sheath by neonatal day 8. This study shows that there are heterogeneous macrophage populations and that HS cells can show various cytoskeletal proteins in rat hepatogenesis.

Immunohistochemical Characterization of Macrophages and Myofibroblasts in α-Naphthylisothiocyanate (ANIT)–Induced Bile Duct Injury and Subsequent Fibrogenesis in Rats

To investigate pathogenesis of post–bile duct (BD) injury fibrosis, interlobular BD epithelial injury was induced in male F344 rats by a single IP injection of α-naphthylisothiocyanate (75 mg/kg body weight) and rats were observed for 12 days. On days 1 to 2, cholangiocytes were injured and desquamated. On days 3 to 5, the affected BD began to regenerate, showing positive staining for CK19 and vimentin. On days 5 to 9, fibrotic areas gradually developed around regenerating BD in Glisson’s sheath. These consisted of cells positive for vimentin, desmin, and α-SMA; vimentin- and desmin-positive cells were increased in early stage (days 1–3), whereas α-SMA-positive cells appeared in mid (days 4–7) and late stages (days 8–12), although there were cells coexpressing these cytoskeletons. On day 12, BD regeneration almost completed, with reduced fibrosis. Macrophages positive for ED2 (CD163) increased transiently in early stage, whereas those reacting to ED1 (CD68), OX6 (MHC II), and SRA-E5 (CD204) showed a consistent increase throughout the experiment. Interestingly, OX6-positive cells were limited to Glisson’s sheath, whereas SRA-E5-positive cells were seen exclusively along sinusoids of hepatic lobules. MCP-1 mRNA increased significantly in early stage. This study shows that macrophages exhibiting different immunophenotypes and distributions participate in post-BD injury fibrosis associated with myofibroblasts expressing various mesenchymal cytoskeletons.

Novel Cholix Toxin Variants, ADP-Ribosylating Toxins in Vibrio cholerae Non-O1/Non-O139 Strains, and Their Pathogenicity

ABSTRACT Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

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.

Immunophenotypical characterization and influence on liver homeostasis of depleting and repopulating hepatic macrophages in rats injected with clodronate

Hepatic macrophages (including Kupffer cells) play a crucial role in the homeostasis and act as mediators of inflammatory response in the liver. Hepatic macrophages were depleted in male F344 rats by a single intravenous injection of liposomal clodronate (CLD; 50mg/kg body weight), and immunophenotypical characteristics of depleting and repopulating macrophages were analyzed by different antibodies specific for macrophages. CD163(+) Kupffer cells were almost completely depleted on post-injection (PI) days 1-12. Macrophages reacting to CD68, Iba-1, and Gal-3 were drastically reduced in number on PI day 1 and then recovered gradually until PI day 12. MHC class II(+) and CD204(+) macrophages were moderately decreased during the observation period. Although hepatic macrophages detectable by different antibodies were reduced in varying degrees, Kupffer cells were the most susceptible to CLD. Liver situation influenced by depleted hepatic macrophages was also investigated. No marked histological changes were seen in the liver, but the proliferating activity of hepatocytes was significantly increased, supported by changes of gene profiles relating to cell proliferation on microarray analysis on PI day 1; the values of AST and ALT were significantly elevated; macrophage induction/activation factors (such as MCP-1, CSF-1, IL-6 and IL-4) were increased exclusively on PI day 1, whereas anti-inflammatory factors such as IL-10 and TGF-β1 remained significantly decreased after macrophage depletion. The present study confirmed importance of hepatic macrophages in liver homeostasis. The condition of hepatic macrophages should be taken into consideration when chemicals capable of inhibiting macrophage functions are evaluated.

Depletion of Hepatic Macrophages Aggravates Liver Lesions Induced in Rats by Thioacetamide (TAA).

Hepatic macrophages play crucial roles in hepatotoxicity. We investigated immunophenotypes of macrophages in liver injury induced in rats by thioacetamide (TAA; 300 mg/kg, intraperitoneal) after hepatic macrophage depletion; hepatic macrophages were depleted by liposomal clodronate (CLD; 10 ml/kg, i.v.) one day before TAA injection. Samples were obtained on post-TAA injection days 0, 1, 2, 3, 5, and 7. TAA injection induced coagulation necrosis of hepatocytes on days 1 through 3 and subsequent reparative fibrosis on days 5 and 7 in the centrilobular area, accompanied by increased numbers of M1 macrophages (expressing cluster of differentiation [CD]68 and major histocompatibility complex class II) and M2 macrophages (expressing CD163 and CD204) mainly on days 1 through 3. TAA + CLD treatment markedly decreased the numbers of M1 and M2 macrophages mainly on days 1 through 3; CD163+ Kupffer cells were most sensitive to CLD depletion. In TAA + CLD–treated rats, interestingly, coagulation necrosis of hepatocytes was prolonged with more increased levels of hepatic enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase) to TAA-treated rats; reparative fibrosis was incomplete and replaced by dystrophic calcification in the injured area, indicating the aggravated damage. Furthermore, in TAA + CLD–treated rats, inflammatory factors (monocyte chemoattractant protein [MCP]-1, interferon-γ, tumor necrosis factor-α, and interleukin-10) and fibrosis-related factors (transforming growth factor-β1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1) were decreased at messenger RNA levels, indicating abnormal macrophage functions. It was clearly demonstrated that hepatic macrophages have important roles in tissue damage and remodeling in hepatotoxicity.

Inflammatory regulation of iron metabolism during thioacetamide-induced acute liver injury in rats

Systemic iron homeostasis is tightly regulated by the interaction between iron regulatory molecules, mainly produced by the liver. However, the molecular mechanisms of iron regulation in liver diseases remain to be elucidated. Here we analyzed the expression profiles of iron regulatory molecules during transient iron overload in a rat model of thioacetamide (TAA)-induced acute liver injury. After TAA treatment, mild hepatocellular degeneration and extensive necrosis were observed in the centrilobular region at hour 10 and on day 1, respectively. Serum iron increased transiently at hour 10 and on day 1, in contrast to hypoferremia in other rodent models of acute inflammation reported previously. Thereafter, up-regulation of hepcidin, a central regulator of systemic iron homeostasis, was observed in hepatocytes on day 2. Expression of transferrin receptor 1 and ferritin subunits increased to a peak on day 3, followed by increases in liver iron content and stainable iron on day 5, in parallel with regeneration of hepatocytes. Histopathological lesions and hepatocellular iron accumulation disappeared until day 10. The hepcidin induction was preceded by activation of IL6/STAT3 pathway, whereas other pathways known to induce hepcidin were down-regulated. IL6 was expressed by MHC class II-positive macrophages in the portal area, suggestive of dendritic cells. Our results suggest that IL6 released by portal macrophages may regulate hepatocyte hepcidin expression via STAT3 activation during transient iron overload in TAA-induced acute liver injury.